2′-C-methyl-3′-O-L-valine ester ribofuranosyl cytidine for treatment of flaviviridae infections

ABSTRACT

The 3′-L-valine ester of β-D-2′-C-methyl-ribofuranosyl cytidine provides superior results against flaviviruses and pestiviruses, including hepatitis C virus. Based on this discovery, compounds, compositions, methods and uses are provided for the treatment of flaviviridae, including HCV, that include the administration of an effective amount of val-mCyd or its salt, ester, prodrug or derivative, optionally in a pharmaceutically acceptable carrier. In an alternative embodiment, val-mCyd is used to treat any virus that replicates through an RNA-dependent RNA polymerase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/607,909, filed Jun. 27, 2003, now U.S. Pat. No. 7,456,155, whichclaims the benefit of priority to U.S. Provisional application No.60/392,351, filed Jun. 28, 2002, the disclosure of which is incorporatedherein by reference.

PARTIES TO A JOINT RESEARCH AGREEMENT

The subject matter of this application arises in part from a jointresearch agreement between Idenix Pharmaceuticals, Inc., UniversitaDegli Studi di Cagliari, Centre National de la Recherche Scientifique,and L'Université Montpellier II.

FIELD OF THE INVENTION

This invention is in the area of pharmaceutical chemistry and, inparticular, is 2′-C-methyl-ribofuranosyl cytidine-3′-O-L-valine esterand pharmaceutically acceptable salts, derivatives and prodrugs thereof,their syntheses and their uses as anti-Flaviviridae agents in thetreatment of hosts, notably humans, infected with a Flaviviridae, and inparticular, hepatitis C, virus.

BACKGROUND OF THE INVENTION

Flaviviridae Viruses

The Flaviviridae family of viruses comprises at least three distinctgenera: pestiviruses, which cause disease in cattle and pigs;flaviviruses, which are the primary cause of diseases such as denguefever and yellow fever; and hepaciviruses, whose sole member is HCV. Theflavivirus genus includes more than 68 members separated into groups onthe basis of serological relatedness (Calisher et al., J. Gen. Virol,1993, 70, 37-43). Clinical symptoms vary and include fever, encephalitisand hemorrhagic fever (Fields Virology, Editors: Fields, B. N., Knipe,D. M., and Howley, P. M., Lippincott-Raven Publishers, Philadelphia,Pa., 1996, Chapter 31, 931-959). Flaviviruses of global concern that areassociated with human disease include the dengue hemorrhagic feverviruses (DHF), yellow fever virus, shock syndrome and Japaneseencephalitis virus (Halstead, S. B., Rev. Infect. Dis., 1984, 6,251-264; Halstead, S. B., Science, 239:476-481, 1988; Monath, T. P., NewEng. J. Med, 1988, 319, 641-643).

The pestivirus genus includes bovine viral diarrhea virus (BVDV),classical swine fever virus (CSFV, also called hog cholera virus) andborder disease virus (BDV) of sheep (Moennig, V. et al. Adv. Vir. Res.1992, 41, 53-98). Pestivirus infections of domesticated livestock(cattle, pigs and sheep) cause significant economic losses worldwide.BVDV causes mucosal disease in cattle and is of significant economicimportance to the livestock industry (Meyers, G. and Thiel, H.-J.,Advances in Virus Research, 1996, 47, 53-118; Moennig V., et al, Adv.Vir. Res. 1992, 41, 53-98). Human pestiviruses have not been asextensively characterized as the animal pestiviruses. However,serological surveys indicate considerable pestivirus exposure in humans.

Pestiviruses and hepaciviruses are closely related virus groups withinthe Flaviviridae family. Other closely related viruses in this familyinclude the GB virus A, GB virus A-like agents, GB virus-B and GBvirus-C (also called hepatitis G virus, HGV). The hepacivirus group(hepatitis C virus; HCV) consists of a number of closely related butgenotypically distinguishable viruses that infect humans. There areapproximately 6 HCV genotypes and more than 50 subtypes. Due to thesimilarities between pestiviruses and hepaciviruses, combined with thepoor ability of hepaciviruses to grow efficiently in cell culture,bovine viral diarrhea virus (BVDV) is often used as a surrogate to studythe HCV virus.

The genetic organization of pestiviruses and hepaciviruses is verysimilar. These positive stranded RNA viruses possess a single large openreading frame (ORF) encoding all the viral proteins necessary for virusreplication. These proteins are expressed as a polyprotein that is co-and post-translationally processed by both cellular and virus-encodedproteinases to yield the mature viral proteins. The viral proteinsresponsible for the replication of the viral genome RNA are locatedwithin approximately the carboxy-terminal. Two-thirds of the ORF aretermed nonstructural (NS) proteins. The genetic organization andpolyprotein processing of the nonstructural protein portion of the ORFfor pestiviruses and hepaciviruses is very similar. For both thepestiviruses and hepaciviruses, the mature nonstructural (NS) proteins,in sequential order from the amino-terminus of the nonstructural proteincoding region to the carboxy-terminus of the ORF, consist of p7, NS2,NS3, NS4A, NS4B, NS5A, and NS5B.

The NS proteins of pestiviruses and hepaciviruses share sequence domainsthat are characteristic of specific protein functions. For example, theNS3 proteins of viruses in both groups possess amino acid sequencemotifs characteristic of serine proteinases and of helicases (Gorbalenyaet al. (1988) Nature 333:22; Bazan and Fletterick (1989) Virology171:637-639; Gorbalenya et al. (1989) Nucleic Acid Res. 17.3889-3897).Similarly, the NSSB proteins of pestiviruses and hepaciviruses have themotifs characteristic of RNA-directed RNA polymerases (Koonin, E. V. andDoija, V. V. (1993) Crit. Rev. Biochem. Molec. Biol. 28:375-430).

The actual roles and functions of the NS proteins of pestiviruses andhepaciviruses in the lifecycle of the viruses are directly analogous. Inboth cases, the NS3 serine proteinase is responsible for all proteolyticprocessing of polyprotein precursors downstream of its position in theORF (Wiskerchen and Collett (1991) Virology 184:341-350; Bartenschlageret al. (1993) J. Virol. 67:3835-3844; Eckart et al. (1993) Biochem.Biophys. Res. Comm. 192:399-406; Grakoui et al. (1993) J. Virol.67:2832-2843; Grakoui et al. (1993) Proc. Natl. Acad. Sci. USA90:10583-10587; Hijikata et al. (1993) J. Virol. 67:4665-4675; Tome etal. (1993) J. Virol. 67:4017-4026). The NS4A protein, in both cases,acts as a cofactor with the NS3 serine protease (Bartenschlager et al.(1994) J. Virol. 68:5045-5055; Failla et al. (1994) J. Virol. 68:3753-3760; Lin et al. (1994) 68:8147-8157; Xu et al. (1997) J. Virol.71:5312-5322). The NS3 protein of both viruses also functions as ahelicase (Kim et al. (1995) Biochem. Biophys. Res. Comm. 215: 160-166;Jin and Peterson (1995) Arch. Biochem. Biophys., 323:47-53; Warrener andCollett (1995) J. Virol. 69:1720-1726). Finally, the NS5B proteins ofpestiviruses and hepaciviruses have the predicted RNA-directed RNApolymerases activity (Behrens et al. (1996) EMBO J. 15:12-22; Lchmann etal. (1997) J. Virol. 71:8416-8428; Yuan et al. (1997) Biochem. Biophys.Res. Comm. 232:231-235; Hagedom, PCT WO 97/12033; Zhong et al. (1998) J.Virol. 72.9365-9369).

Hepatitis C Virus

The hepatitis C virus (HCV) is the leading cause of chronic liverdisease worldwide. (Boyer, N. et al. J. Hepatol. 32:98-112, 2000). HCVcauses a slow growing viral infection and is the major cause ofcirrhosis and hepatocellular carcinoma (Di Besceglie, A. M. and Bacon,B. R., Scientific American, Oct.: 80-85, (1999); Boyer, N. et al. J.Hepatol. 32:98-112, 2000). An estimated 170 million persons are infectedwith HCV worldwide. (Boyer, N. et al. J. Hepatol. 32:98-112, 2000).Cirrhosis caused by chronic hepatitis C infection accounts for8,000-12,000 deaths per year in the United States, and HCV infection isthe leading indication for liver transplantation.

HCV is known to cause at least 80% of posttransfusion hepatitis and asubstantial proportion of sporadic acute hepatitis. Preliminary evidencealso implicates HCV in many cases of “idiopathic” chronic hepatitis,“cryptogenic” cirrhosis, and probably hepatocellular carcinoma unrelatedto other hepatitis viruses, such as Hepatitis B Virus (HBV). A smallproportion of healthy persons appear to be chronic HCV carriers, varyingwith geography and other epidemiological factors. The numbers maysubstantially exceed those for HBV, though information is stillpreliminary; how many of these persons have subclinical chronic liverdisease is unclear. (The Merck Manual, ch. 69, p. 901, 16th ed.,(1992)).

HCV is an enveloped virus containing a positive-sense single-strandedRNA genome of approximately 9.4 kb. The viral genome consists of a 5′untranslated region (UTR), a long open reading frame encoding apolyprotein precursor of approximately 3011 amino acids, and a short 3′UTR. The 5′ UTR is the most highly conserved part of the HCV genome andis important for the initiation and control of polyprotein translation.Translation of the HCV genome is initiated by a cap-independentmechanism known as internal ribosome entry. This mechanism involves thebinding of ribosomes to an RNA sequence known as the internal ribosomeentry site (IRES). An RNA pseudoknot structure has recently beendetermined to be an essential structural element of the HCV IRES. Viralstructural proteins include a nucleocapsid core protein (C) and twoenvelope glycoproteins, E1 and E2. HCV also encodes two proteinases, azinc-dependent metalloproteinase encoded by the NS2-NS3 region and aserine proteinase encoded in the NS3 region. These proteinases arerequired for cleavage of specific regions of the precursor polyproteininto mature peptides. The carboxyl half of nonstructural protein 5,NS5B, contains the RNA-dependent RNA polymerase. The function of theremaining nonstructural proteins, NS4A and NS4B, and that of NS5A (theamino-terminal half of nonstructural protein 5) remain unknown.

A significant focus of current antiviral research is directed to thedevelopment of improved methods of treatment of chronic HCV infectionsin humans (Di Besceglie, A. M. and Bacon, B. R., Scientific American,October: 80-85, (1999)).

Treatment of HCV Infection with Interferon

Interferons (IFNs) have been commercially available for the treatment ofchronic hepatitis for nearly a decade. IFNs are glycoproteins producedby immune cells in response to viral infection. IFNs inhibit replicationof a number of viruses, including HCV, and when used as the soletreatment for hepatitis C infection, IFN can in certain cases suppressserum HCV-RNA to undetectable levels. Additionally, IFN can normalizeserum amino transferase levels. Unfortunately, the effect of IFN istemporary and a sustained response occurs in only 8%-9% of patientschronically infected with HCV (Gary L. Davis. Gastroenterology118:S104-S114, 2000). Most patients, however, have difficulty toleratinginterferon treatment, which causes severe flu-like symptoms, weightloss, and lack of energy and stamina.

A number of patents disclose Flaviviridae, including HCV, treatments,using interferon-based therapies. For example, U.S. Pat. No. 5,980,884to Blatt et al. discloses methods for retreatment of patients afflictedwith HCV using consensus interferon. U.S. Pat. No. 5,942,223 to Bazer etal. discloses an anti-HCV therapy using ovine or bovine interferon-tau.U.S. Pat. No. 5,928,636 to Alber et al. discloses the combinationtherapy of interleukin-12 and interferon alpha for the treatment ofinfectious diseases including HCV. U.S. Pat. No. 5,849,696 to Chretienet al. discloses the use of thymosins, alone or in combination withinterferon, for treating HCV. U.S. Pat. No. 5,830,455 to Valtuena et al.discloses a combination HCV therapy employing interferon and a freeradical scavenger. U.S. Pat. No. 5,738,845 to Imakawa discloses the useof human interferon tau proteins for treating HCV. Otherinterferon-based treatments for HCV are disclosed in U.S. Pat. No.5,676,942 to Testa et al., U.S. Pat. No. 5,372,808 to Blatt et al., andU.S. Pat. No. 5,849,696. A number of patents also disclose pegylatedforms of interferon, such as, U.S. Pat. Nos. 5,747,646, 5,792,834 and5,834,594 to Hoffmann-La Roche Inc; PCT Publication No. WO 99/32139 andWO 99/32140 to Enzon; WO 95/13090 and U.S. Pat. Nos. 5,738,846 and5,711,944 to Schering; and U.S. Pat. No. 5,908,621 to Glue et al.

Interferon alpha-2a and interferon alpha-2b are currently approved asmonotherapy for the treatment of HCV. ROFERON®-A (Roche) is therecombinant form of interferon alpha-2a. PEGASYS® (Roche) is thepegylated (i.e. polyethylene glycol modified) form of interferonalpha-2a. INTRON®A (Schering Corporation) is the recombinant form ofInterferon alpha-2b, and PEG-INTRON® (Schering Corporation) is thepegylated form of interferon alpha-2b.

Other forms of interferon alpha, as well as interferon beta, gamma, tauand omega are currently in clinical development for the treatment ofHCV. For example, INFERGEN (interferon alphacon-1) by InterMune,OMNIFERON (natural interferon) by Viragen, ALBUFERON by Human GenomeSciences, REBIF (interferon beta-1a) by Ares-Serono, Omega Interferon byBioMedicine, Oral Interferon Alpha by Amarillo Biosciences, andinterferon gamma, interferon tau, and interferon gamma-1b by InterMuneare in development.

Ribivarin

Ribavirin (1-β-D-ribofuranosyl-1-1,2,4-triazole-3-carboxamide) is asynthetic, non-interferon-inducing, broad spectrum antiviral nucleosideanalog sold under the trade name, Virazole (The Merck Index, 11thedition, Editor: Budavari, S., Merck & Co., Inc., Rahway, N.J., p 1304,1989). U.S. Pat. No. 3,798,209 and RE29,835 disclose and claimribavirin. Ribavirin is structurally similar to guanosine, and has invitro activity against several DNA and RNA viruses includingFlaviviridae (Gary L. Davis. Gastroenterology 118:S104-S114, 2000).

Ribavirin reduces serum amino transferase levels to normal in 40% ofpatients, but it does not lower serum levels of HCV-RNA (Gary L. Davis.Gastroenterology 118:S104-S114, 2000). Thus, ribavirin alone is noteffective in reducing viral RNA levels. Additionally, ribavirin hassignificant toxicity and is known to induce anemia.

Ribavirin is not approved fro monotherapy against HCV. It has beenapproved in combination with interferon alpha-2a or interferon alpha-2bfor the treatment of HCV.

Combination of Interferon and Ribavirin

The current standard of care for chronic hepatitis C is combinationtherapy with an alpha interferon and ribavirin. The combination ofinterferon and ribavirin for the treatment of HCV infection has beenreported to be effective in the treatment of interferon naïve patients(Battaglia, A. M. et al., Ann. Pharmacother. 34:487-494, 2000), as wellas for treatment of patients when histological disease is present(Berenguer, M. et al. Antivir. Ther. 3(Suppl. 3):125-136, 1998). Studieshave show that more patients with hepatitis C respond to pegylatedinterferon-alpha/ribavirin combination therapy than to combinationtherapy with unpegylated interferon alpha. However, as with monotherapy,significant side effects develop during combination therapy, includinghemolysis, flu-like symptoms, anemia, and fatigue. (Gary L. Davis.Gastroenterology 118:S104-S114, 2000).

Combination therapy with PEG-INTRON® (peginterferon alpha-2b) andREBETOL® (Ribavirin, USP) Capsules is available from ScheringCorporation. REBETOL® (Schering Corporation) has also been approved incombination with INTRON® A (Interferon alpha-2b, recombinant, ScheringCorporation). Roche's PEGASYS® (pegylated interferon alpha-2a) andCOPEGUS® (ribavirin) are also approved for the treatment of HCV.

PCT Publication Nos. WO 99/59621, WO 00/37110, WO 01/81359, WO 02/32414and WO 03/024461 by Schering Corporation disclose the use of pegylatedinterferon alpha and ribavirin combination therapy for the treatment ofHCV. PCT Publication Nos. WO 99/15194, WO 99/64016, and WO 00/24355 byHoffmann-La Roche Inc also disclose the use of pegylated interferonalpha and ribavirin combination therapy for the treatment of HCV.

Additional Methods to Treat Flaviviridae Infections

The development of new antiviral agents for flaviviridae infections,especially hepatitis C, is currently underway. Specific inhibitors ofHCV-derived enzymes such as protease, helicase, and polymeraseinhibitors are being developed. Drugs that inhibit other steps in HCVreplication are also in development, for example, drugs that blockproduction of HCV antigens from the RNA (IRES inhibitors), drugs thatprevent the normal processing of HCV proteins (inhibitors ofglycosylation), drugs that block entry of HCV into cells (by blockingits receptor) and nonspecific cytoprotective agents that block cellinjury caused by the virus infection. Further, molecular approaches arealso being developed to treat hepatitis C, for example, ribozymes, whichare enzymes that break down specific viral RNA molecules, and antisenseoligonucleotides, which are small complementary segments of DNA thatbind to viral RNA and inhibit viral replication, are underinvestigation. A number of HCV treatments are reviewed by Bymock et al.in Antiviral Chemistry & Chemotherapy, 11:2; 79-95 (2000) and DeFrancesco et al. in Antiviral Research, 58: 1-16 (2003).

Examples of classes of drugs that are being developed to treatFlaviviridae infections include:

(1) Protease Inhibitors

Substrate-based NS3 protease inhibitors (Attwood et al, Antiviralpeptide derivatives, PCT WO 98/22496, 1998; Attwood et al., AntiviralChemistry and Chemotherapy 1999, 10, 259-273; Attwood et al, Preparationand use of amino acid derivatives as anti-viral agents, German PatentPub. DE 19914474; Tung et al. Inhibitors of serine proteases,particularly hepatitis C virus NS3 protease, PCT WO 98/17679), includingalphaketoamides and hydrazinoureas, and inhibitors that terminate in anelectrophile such as a boronic acid or phosphonate (Llinas-Brunet et al,Hepatitis C inhibitor peptide analogues, PCT WO 99/07734) are beinginvestigated.

Non-substrate-based NS3 protease inhibitors such as2,4,6-trihydroxy-3-nitro-benzamide derivatives (Sudo K. et al,Biochemical and Biophysical Research Communications, 1997, 238, 643-647;Sudo K. et al. Antiviral Chemistry and Chemotherapy, 1998, 9, 186),including RD3-4082 and RD3-4078, the former substituted on the amidewith a 14 carbon chain and the latter processing a para-phenoxyphenylgroup are also being investigated.

Sch 68631, a phenanthrenequinone, is an HCV protease inhibitor (Chu M.et al., Tetrahedron Letters 37:7229-7232, 1996). In another example bythe same authors, Sch 351633, isolated from the fungus Penicilliumgriseofulvum, was identified as a protease inhibitor (Chu M. et al.,Bioorganic and Medicinal Chemistry Letters 9:1949-1952). Nanomolarpotency against the HCV NS3 protease enzyme has been achieved by thedesign of selective inhibitors based on the macromolecule eglin c. Eglinc, isolated from leech, is a potent inhibitor of several serineproteases such as S. griseus proteases A and B, α-chymotrypsin, chymaseand subtilisin. Qasim M. A. et al., Biochemistry 36:1598-1607, 1997.

Several U.S. patents disclose protease inhibitors for the treatment ofHCV. For example, U.S. Pat. No. 6,004,933 to Spruce et al. discloses aclass of cysteine protease inhibitors for inhibiting HCV endopeptidase2. U.S. Pat. No. 5,990,276 to Zhang et al. discloses syntheticinhibitors of hepatitis C virus NS3 protease. The inhibitor is asubsequence of a substrate of the NS3 protease or a substrate of theNS4A cofactor. The use of restriction enzymes to treat HCV is disclosedin U.S. Pat. No. 5,538,865 to Reyes et al. Peptides as NS3 serineprotease inhibitors of HCV are disclosed in WO 02/008251 to CorvasInternational, Inc, and WO 02/08187 and WO 02/008256 to ScheringCorporation. HCV inhibitor tripeptides are disclosed in U.S. Pat. Nos.6,534,523, 6,410,531, and 6,420,380 to Boehringer Ingelheim and WO02/060926 to Bristol Myers Squibb. Diaryl peptides as NS3 serineprotease inhibitors of HCV are disclosed in WO 02/48172 to ScheringCorporation. Imidazoleidinones as NS3 serine protease inhibitors of HCVare disclosed in WO 02/08198 to Schering Corporation and WO 02/48157 toBristol Myers Squibb. WO 98/17679 to Vertex Pharmaceuticals and WO02/48116 to Bristol Myers Squibb also disclose HCV protease inhibitors.

-   -   (2) Thiazolidine derivatives which show relevant inhibition in a        reverse-phase HPLC assay with an NS3/4A fusion protein and        NS5A/5B substrate (Sudo K. et al., Antiviral Research, 1996, 32,        9-18), especially compound RD-1-6250, possessing a fused        cinnamoyl moiety substituted with a long alkyl chain, RD4 6205        and RD4 6193;    -   (3) Thiazolidines and benzanilides identified in Kakiuchi N. et        al. J. EBS Letters 421, 217-220; Takeshita N. et al. Analytical        Biochemistry, 1997, 247, 242-246;    -   (4) A phenan-threnequinone possessing activity against protease        in a SDS-PAGE and autoradiography assay isolated from the        fermentation culture broth of Streptomyces sp., Sch 68631        (Chu M. et al., Tetrahedron Letters, 1996, 37, 7229-7232), and        Sch 351633, isolated from the fungus Penicillium griseofulvum,        which demonstrates activity in a scintillation proximity assay        (Chu M. et al., Bioorganic and Medicinal Chemistry Letters 9,        1949-1952);    -   (5) Helicase inhibitors (Diana G. D. et al, Compounds,        compositions and methods for treatment of hepatitis C, U.S. Pat.        No. 5,633,358; Diana G. D. et al., Piperidine derivatives,        pharmaceutical compositions thereof and their use in the        treatment of hepatitis C, PCT WO 97/36554);    -   (6) Nucleotide polymerase inhibitors and gliotoxin (Ferrari R.        et al. Journal of Virology, 1999, 73, 1649-1654), and the        natural product cerulenin (Lohmann V. et al., Virology, 1998,        249, 108-118);    -   (7) Antisense phosphorothioate oligodeoxynucleotides (S-ODN)        complementary to sequence stretches in the 5′ non-coding region        (NCR) of the virus (Alt M. et al, Hepatology, 1995, 22,        707-717), or nucleotides 326-348 comprising the 3′ end of the        NCR and nucleotides 371-388 located in the core coding region of        the HCV RNA (Alt M. et al., Archives of Virology, 1997, 142,        589-599; Galderisi U. et al., Journal of Cellular Physiology,        1999, 181, 251-257);    -   (8) Inhibitors of IRES-dependent translation (Ikeda N et al.,        Agent for the prevention and treatment of hepatitis C, Japanese        Patent Pub. JP-08268890; Kai Y. et al. Prevention and treatment        of viral diseases, Japanese Patent Pub. JP-10101591);    -   (9) Ribozymes, such as nuclease-resistant ribozymes        (Maccjak, D. J. et al., Hepatology 1999, 30, abstract 995) and        those disclosed in U.S. Pat. No. 6,043,077 to Barber et al., and        U.S. Pat. Nos. 5,869,253 and 5,610,054 to Draper et al.; and    -   (10) Nucleoside analogs have also been developed for the        treatment of Flaviviridae infections.

Idenix Pharmaceuticals disclosed the use of branched nucleosides in thetreatment of flaviviruses (including HCV) and pestiviruses inInternational Publication Nos. WO 01/90121 and WO 01/92282.Specifically, a method for the treatment of hepatitis C infection (andflaviviruses and pestiviruses) in humans and other host animals isdisclosed in the Idenix publications that includes administering aneffective amount of a biologically active 1′, 2′, 3′ or 4′-branched β-Dor β-L nucleosides or a pharmaceutically acceptable salt or derivativethereof, administered either alone or in combination with anotherantiviral agent, optionally in a pharmaceutically acceptable carrier.

Other patent applications disclosing the use of certain nucleosideanalogs to treat hepatitis C virus include: PCT/CA00/01316 (WO 01/32153;filed Nov. 3, 2000) and PCT/CA01/00197 (WO 01/60315; filed Feb. 19,2001) filed by BioChem Pharma, Inc. (now Shire Biochem, Inc.);PCT/US02/01531 (WO 02/057425; filed Jan. 18, 2002) and PCT/US02/03086(WO 02/057287; filed Jan. 18, 2002) filed by Merck & Co., Inc.,PCT/EP01/09633 (WO 02/18404; published Aug. 21, 2001) filed by Roche,and PCT Publication Nos. WO 01/79246 (filed Apr. 13, 2001), WO 02/32920(filed Oct. 18, 2001) and WO 02/48165 by Pharmasset, Ltd.

PCT Publication No. WO 99/43691 to Emory University, entitled“2′-Fluoronucleosides” discloses the use of certain 2′-fluoronucleosidesto treat HCV.

Eldrup et al. (Oral Session V, Hepatitis C Virus, Flaviviridae; 16^(th)International Conference on Antiviral Research (Apr. 27, 2003, Savannah,Ga.)) described the structure activity relationship of 2′-modifiednucleosides for inhibition of HCV.

Bhat et al. (Oral Session V, Hepatitis C Virus, Flaviviridae, 2003 (OralSession V, Hepatitis C Virus, Flaviviridae; 16^(th) InternationalConference on Antiviral Research (Apr. 27, 2003, Savannah, Ga.); p A75)described the synthesis and pharmacokinetic properties of nucleosideanalogues as possible inhibitors of HCV RNA replication. The authorsreport that 2′-modified nucleosides demonstrate potent inhibitoryactivity in cell-based replicon assays.

Olsen et al. (Oral Session V, Hepatitis C Virus, Flaviviridae; 16^(th)International Conference on Antiviral Research (Apr. 27, 2003, Savannah,Ga.) p A76) also described the effects of the 2′-modified nucleosides onHCV RNA replication.

-   -   (11) Other miscellaneous compounds including        1-amino-alkylcyclohexanes (U.S. Pat. No. 6,034,134 to Gold et        al.), alkyl lipids (U.S. Pat. No. 5,922,757 to Chojkier et al.),        vitamin E and other antioxidants (U.S. Pat. No. 5,922,757 to        Chojkier et al.), squalene, amantadine, bile acids (U.S. Pat.        No. 5,846,964 to Ozeki et al.), N-(phosphonoacetyl)-L-aspartic        acid, (U.S. Pat. No. 5,830,905 to Diana et al.),        benzenedicarboxamides (U.S. Pat. No. 5,633,388 to Diana et al.),        polyadenylic acid derivatives (U.S. Pat. No. 5,496,546 to Wang        et al.), 2′,3′-dideoxyinosine (U.S. Pat. No. 5,026,687 to        Yarchoan et al.), benzimidazoles (U.S. Pat. No. 5,891,874 to        Colacino et al.), plant extracts (U.S. Pat. No. 5,837,257 to        Tsai et al., U.S. Pat. No. 5,725,859 to Omer et al., and U.S.        Pat. No. 6,056,961), and piperidenes (U.S. Pat. No. 5,830,905 to        Diana et al.).    -   (12) Other compounds currently in preclinical or clinical        development for treatment of hepatitis C virus include:        Interleukin-10 by Schering-Plough, IP-501 by Interneuron,        Merimebodib (VX-497) by Vertex, AMANTADINE® (Symmetrel) by Endo        Labs Solvay, HEPTAZYME® by RPI, IDN-6556 by Idun Pharma.,        XTL-002 by XTL., HCV/MF59 by Chiron, CIVACIR® (Hepatitis C        Immune Globulin) by NABI, LEVOVIRINS by ICN/Ribapharm,        VIRAMIDINE® by ICN/Ribapharm, ZADAXIN® (thymosin alpha-1) by Sci        Clone, thymosin plus pegylated interferon by Sci Clone, CEPLENE®        (histamine dihydrochloride) by Maxim, VX 950/LY 570310 by        Vertex/Eli Lilly, ISIS 14803 by Isis Pharmaceutical/Elan,        IDN-6556 by Idun Pharmaceuticals, Inc., JTK 003 by AKROS Pharma,        BILN-2061 by Boehringer Ingelheim, CellCept (mycophenolate        mofetil) by Roche, T67, a β-tubulin inhibitor, by Tularik, a        therapeutic vaccine directed to E2 by Innogenetics, FK788 by        Fujisawa Healthcare, Inc., 1 dB 1016 (Siliphos, oral        silybin-phosphatdylcholine phytosome), RNA replication        inhibitors (VP50406) by ViroPharma/Wyeth, therapeutic vaccine by        Intercell, therapeutic vaccine by Epimmune/Genencor, IRES        inhibitor by Anadys, ANA 245 and ANA 246 by Anadys,        immunotherapy (Therapore) by Avant, protease inhibitor by        Corvas/SChering, helicase inhibitor by Vertex, fusion inhibitor        by Trimeris, T cell therapy by CellExSys, polymerase inhibitor        by Biocryst, targeted RNA chemistry by PTC Therapeutics,        Dication by Immtech, Int., protease inhibitor by Agouron,        protease inhibitor by Chiron/Medivir, antisense therapy by AVI        BioPharma, antisense therapy by Hybridon, hemopurifier by        Aethlon Medical, therapeutic vaccine by Merix, protease        inhibitor by Bristol-Myers Squibb/Axys, Chron-VacC, a        therapeutic vaccine, by Tripep, UT 231B by United Therapeutics,        protease, helicase and polymerase inhibitors by Genelabs        Technologies, IRES inhibitors by Immusol, R803 by Rigel        Pharmaceuticals, INFERGEN® (interferon alphacon-1) by InterMune,        OMNIFERON® (natural interferon) by Viragen, ALBUFERON® by Human        Genome Sciences, REBIF (interferon beta-1a) by Ares-Serono,        Omega Interferon by BioMedicine, Oral Interferon Alpha by        Amarillo Biosciences, interferon gamma, interferon tau, and        Interferon gamma-1b by InterMune.

Nucleoside prodrugs have been previously described for the treatment ofother forms of hepatitis. WO 01/96353 (filed Jun. 15, 2001) to IdenixPharmaceuticals, discloses 2′-deoxy-β-L-nucleosides and their3′-prodrugs for the treatment of HBV. U.S. Pat. No. 4,957,924 toBeauchamp discloses various therapeutic esters of acyclovir.

In light of the fact that HCV infection has reached epidemic levelsworldwide, and has tragic effects on the infected patient, there remainsa strong need to provide new effective pharmaceutical agents to treathepatitis C that have low toxicity to the host.

Further, given the rising threat of other flaviviridae infections, thereremains a strong need to provide new effective pharmaceutical agentsthat have low toxicity to the host.

Therefore, it is an object of the present invention to provide acompound, method and composition for the treatment of a host infectedwith hepatitis C virus.

It is another object of the present invention to provide a method andcomposition generally for the treatment of patients infected withpestiviruses, flaviviruses, or hepaciviruses.

SUMMARY OF THE INVENTION

It has been discovered that the 3′-L-valine ester ofβ-D-2′-C-methyl-ribofuranosyl cytidine (referred to alternatively belowas val-mCyd) provides superior results against flaviviruses andpestiviruses, including hepatitis C virus. Based on this discovery,compounds, compositions, methods and uses are provided for the treatmentof flaviviridae, including HCV, that include the administration of aneffective amount of val-mCyd or its salt, ester, prodrug or derivative,optionally in a pharmaceutically acceptable carrier. In an alternativeembodiment, val-mCyd is used to treat any virus that replicates throughan RNA-dependent RNA polymerase.

Therefore, a first embodiment provides a compound of Formula (I),β-D-2′-C-methyl-ribofuranosyl cytidine or a pharmaceutically acceptablesalt thereof, and its uses in medical therapy and in the manufacture ofa medicant to treat hosts, particularly humans, infected with aflavivirus or pestivirus, including HCV.

The compound val-mCyd is converted to the parent MCyd throughde-esterification in the gastrointestinal mucosa, blood or liver, and isactively transported from the gastrointestinal lumen after oral deliveryinto the bloodstream by an amino acid transporter function in the mucosaof the gastrointestinal tract. This accounts for the increase in oralbioavailability compared to the parent 2′-branched nucleoside that istransported primarily by a nucleoside transporter function. There isalso reduced competition with other nucleosides or nucleoside analogsthat are transported by the nucleoside transporter function and not theamino acid transporter function. As partial de-esterification occursprior to complete absorption, the mono or divaline ester continues to beabsorbed using the amino acid transporter function. Therefore, thesuperior outcome of better absorption, bioavailability, and reducedcompetition with other nucleosides or nucleoside analogs for uptake intothe bloodstream is achieved.

In hepatitis C infected chimpanzees, val-mCyd reduced the mean HCV RNAlevel by 0.83 log₁₀ copies/ml (8.3 mg/kg/day) and 1.05 log₁₀ copies/ml(16.6 mg/kg/day) in seven days. The chimps exhibited no drug-relatedsafety issues.

The parent nucleoside (mCyd) framework can exist as a β-D or β-Lnucleoside. In a preferred embodiment, the pharmaceutically acceptablecompound is administered in a form that is at least 90% of the β-Denantiomer. In another embodiment, val-mCyd is at least 95% of the β-Denantiomer. The valine ester also has enantiomeric forms. In a preferredembodiment, the valine moiety is at least 90% of the L-enantiomer. Inanother embodiment, the valine moiety is at least 95% of theL-enantiomer. In alternative embodiments, the compounds are used asracemic mixtures or as any combination of β-D or β-L parent nucleosideand L or D amino acid.

In one specific embodiment, the compound of Formula (I) isβ-D-2′-C-methyl-ribofuranosyl cytidine-3′-O-L-valine ester.HCl salt. Inanother specific embodiment, the compound of Formula (II), theβ-D-2′-C-methyl-ribofuranosyl cytidine dihydrochloride salt, is providedfor administration to a host, particularly a human, infected with aflavivirus or pestivirus infection. In other embodiments, tosylate,methanesulfonate, acetate, citrate, malonate, tartarate, succinate,benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate, formate,fumarate, propionate, glycolate, lactate, pyruvate, oxalate, maleate,salicyate, sulfate, sulfonate, nitrate, bicarbonate, hydrobromate,hydrobromide, hydroiodide, carbonate, and phosphoric acid salts areprovided. A particularly preferred embodiment is the mono ordihydrochloride salt.

In another embodiment, a pharmaceutical composition comprising2′-C-methyl-cytidine-3′-O-L-valine ester or its pharmaceuticallyacceptable salt, including a mono or di HCl salt, ester, prodrug orderivative thereof together with a pharmaceutically acceptable carrier,excipient or diluent is provided.

In an alternative embodiment, the 5′-hydroxyl group is replaced with a5′-OR, wherein R is phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipids; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R is independently Hor phosphate.

The active compounds of the present invention can be administered incombination or alternation with another agent that is active against aflavivirus or pestivirus, including HCV (including any described orreferred to in the Background of the Invention), or other usefulbiological agent. Thus another principal embodiment is a pharmaceuticalcomposition that includes β-D-2′-C-methyl-ribofuranosylcytidine-3′-O-L-valine ester- or a pharmaceutically acceptable salt(including a mono- or di-HCl salt), ester, prodrug or pharmaceuticallyacceptable derivative thereof, together with one or more other effectiveantiviral agents, optionally with a pharmaceutically acceptable carrieror diluent. In another embodiment, a method is provided that includesadministering β-D-2′-C-methyl-ribofuranosyl cytidine-3′-O-L-valine esteror a salt, ester, prodrug or derivative thereof, in combination or inalternation with one or more second antiviral agents, optionally with apharmaceutically acceptable carrier or diluent.

Any second antiviral agent may be selected that imparts the desiredbiological effect. Nonlimiting examples include interferon, ribavirin,an interleukin, an NS3 protease inhibitor, a cysteine proteaseinhibitor, phenanthrenequinone, a thiazolidine derivative, thiazolidine,benzanilide, a helicase inhibitor, a polymerase inhibitor, a nucleotideanalogue, gliotoxin, cerulenin, antisense phosphorothioateoligodeoxynucleotides, inhibitor of IRES-dependent translation, orribozyme. In one particular embodiment, the second antiviral agent isselected from natural interferon, interferon alpha (includinginterferon-alpha-2a and interferon-alpha-2b), interferon beta (includinginterferon beta-1a), omega interferon, interferon gamma (includinginterferon gamma-1b), interferon tau, and consensus interferon. Any ofthese interferons can be stabilized or otherwise modified to improve thetolerance and biological stability or other biological properties. Onecommon modification is pegylation (modification with polyethyleneglycol). In one particular embodiment, the second antiviral agent ispegylated or unpegylated interferon 2-alpha.

In an alternative embodiment, the active compound is a 3′-amino acidester of β-D-2′-C-methyl-ribofuranosyl cytidine, wherein the amino acidcan be natural or synthetic and can be in a D or L stereoconfiguration.In another embodiment, the active compound is a 3′-acyl ester ofβ-D-2′-C-methyl-ribofuranosyl cytidine.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 a and 1 b are illustration of two processes for the preparationof β-D-2′-C-methyl-ribofuranosyl cytidine-3′-O-L-valine esterdihydrochloride, as described in Example 1.

FIG. 2 is a photocopy of a gel illustrating the site-specific chaintermination of in vitro RNA synthesis by β-D-2′-C-methyl-ribofuranosylcytidine triphosphate at specified guanine residues in RNA templates, asdescribed in Example 9.

FIG. 3 is a graph of the titer of bovine viral diarrhea virus (BVDV)over number of passages of BVDV infected MDBK cells, indicatingeradication of a persistent BVDV infection by prolonged treatment withβ-D-2′C-methyl-ribofuranosyl cytidine (16 uM) as described in Example10. Arrows indicate points at which a portion of cells were withdrawnfrom drug treatment.

FIGS. 4 a and 4 b are graphs of the concentration of bovine viraldiarrhea virus (BVDV) in MDBK cells persistently infected with thevirus, as described in Example 11. These graphs indicate the synergybetween β-D-2′-C-methyl-ribofuranosyl cytidine and interferon alpha 2b(IntronA) in reducing the viral titer. FIG. 4 a is a graph of the effectof β-D-2′-C-methyl-ribofuranosyl cytidine and IntronA on BVDV (strainNY-1) titers in persistently infected MDBK cells over time. FIG. 4 b isa graph of the effect of β-D-2′-C-methyl-ribofuranosyl cytidine incombination with IntronA on BVDV (strain I-N-dIns) titers inpersistently-infected MDBK cells.

FIGS. 5 a-d illustrate the results of experiments studying thedevelopment of resistance to β-D-2′-C-methyl-ribofuranosyl cytidinetreated MDBK cells, infected with bovine viral diarrhea virus (BVDV), asdescribed in Example 12. FIG. 5 a is a graph of a representativeexperiment showing the effect over twenty eight days ofβ-D-2′-C-methyl-ribofuranosyl cytidine or IntronA treatment on BVDV(strain I-N-dIns) titers in persistently infected MDBK cells. FIG. 5 bis a photocopy of a dish plated with infected MDBK cells thatillustrates the size of the foci formed by phenotypes of the wild-typeBVDV (strain I-N-dIns), versus the β-D-2′-C-methyl-ribofuranosylcytidine-resistant BVDV (I-N-dIns 107R), indicating that the resistantvirus formed much smaller foci than the wild-type, I-N-dIns strain. FIG.5 c is a graph of the titer of BVDV strains I-N-dIns or I-N-dIns-107Rover hours post-infection in infected MDBK cells. FIG. 5 d is a graph ofthe effect of Intron A on the BVDV viral titer yield in de novo-infectedMDBK cells treated with IntronA.

FIG. 6 is a graph of the concentration of hepatitis C virus (Log₁₀) inindividual chimpanzees over days of treatment withβ-D-2′-C-methyl-ribofuranosyl cytidine-3′-O-L-valine ester as describedin Example 13.

FIG. 7 is a graph of the concentration of hepatitis C virus inindividual chimpanzees over days of treatment withβ-D-2′-C-methyl-ribofuranosyl cytidine-3′-O-L-valine ester as comparedto baseline, as described in Example 13.

FIG. 8 is a graph of percent of total β-D-2′-C-methyl-ribofuranosylcytidine-3′-O-L-valine ester remaining in samples over time afterincubation of the drug in human plasma at 4° C., 21° C., and 37° C., asdescribed in Example 14.

FIG. 9 a is a graph showing the relative levels of the di- andtri-phosphate derivatives of β-D-2′-C-methyl-ribofuranosyl cytidine andβ-D-2′-C-methyl-ribofuranosyl uridine (mUrd) after incubation of HepG2cells with 10 μM β-D-2′-C-methyl-ribofuranosyl cytidine over time, asdescribed in Example 14. FIG. 9 b is a graph of the decay of thetri-phosphate derivative of β-D-2′-C-methyl-ribofuranosyl cytidine afterincubation of HepG2 cells with 10 μM β-D-2′-C-methyl-ribofuranosylcytidine over time. FIG. 9 c is a graph of the concentration of the di-and tri-phosphate derivatives of β-D-2′-C-methyl-ribofuranosyl cytidineand β-D-2′-C-methyl-ribofuranosyl uridine (mUrd) after incubation ofHepG2 cells with 10 μM β-D-2′-C-methyl-ribofuranosyl cytidine atincreasing concentrations of the drug (μM).

FIG. 10 is a graph of the concentration (ng/ml) ofβ-D-2′-C-methyl-ribofuranosyl cytidine in human serum afteradministration of β-D-2′-C-methyl-ribofuranosyl cytidine-3′-O-L-valineester to patients, as described in Example 17.

FIG. 11 is a graph of the median change of the titer of hepatitis Cvirus in human patients after administration ofβ-D-2′-C-methyl-ribofuranosyl cytidine-3′-O-L-valine ester, as describedin Example 17. The graph indicates change from baseline in Log₁₀ HCV RNAby patient visit.

DETAILED DESCRIPTION OF THE INVENTION

It has been discovered that the 3′-L-valine ester ofβ-D-2′-C-methyl-ribofuranosyl cytidine (referred to alternatively belowas val-mCyd) provides superior results against flaviviruses andpestiviruses, including hepatitis C virus. Based on this discovery,compounds, compositions, methods and uses are provided for the treatmentof flaviviridae, including HCV, that include the administration of aneffective amount of val-mCyd or its salt, ester, prodrug or derivative,optionally in a pharmaceutically acceptable carrier. In an alternativeembodiment, val-mCyd is used to treat any virus that replicates throughan RNA-dependent RNA polymerase.

The disclosed compounds or their pharmaceutically acceptable derivativesor salts or pharmaceutically acceptable formulations containing thesecompounds are useful in the prevention and treatment of flaviviridae(including HCV) infections and other related conditions such as anti-HCVantibody positive and HCV-positive conditions, and hepatitis C relatedhepatic cancer (e.g., hepatocellular carcinoma) and hepatic tumors. Inaddition, these compounds or formulations can be used prophylacticallyto prevent or retard the progression of clinical illness in individualswho are anti-HCV (or more generally anti-flaviviridae) antibody orHCV-antigen or flaviviridae-antigen positive, or who have been exposedto HCV or another flaviviridae virus.

In summary, the present invention includes the following features:

-   -   (a) 3′-L-valine ester of β-D-2-C-methyl-ribofuranosyl cytidine,        and pharmaceutically acceptable prodrugs, derivatives and salts        thereof, including specifically the mono- and di-hydrochloride        salts;    -   (b) 3′-L-valine ester of β-D-2′-C-methyl-ribofuranosyl cytidine,        and pharmaceutically acceptable prodrugs, derivatives and salts        thereof for use in medical therapy, for example for the        treatment or prophylaxis of an flaviridae (including HCV)        infection;    -   (c) use of the 3′-L-valine ester of        β-D-2′-C-methyl-ribofuranosyl cytidine, and pharmaceutically        acceptable prodrugs, derivatives and salts thereof in the        manufacture of a medicament for treatment of a flaviviridae        (including HCV) infection;    -   (d) pharmaceutical formulations comprising the 3′-L-valine ester        of β-D-2′-C-methyl-ribofuranosyl cytidine, and pharmaceutically        acceptable prodrugs, derivatives and salts thereof together with        a pharmaceutically acceptable carrier or diluent;    -   (e) processes for the preparation of the 3′-L-valine ester of        β-D-2′-C-methyl-ribofuranosyl cytidine;    -   (f) use of the 3′-L-valine ester of        β-D-2′-C-methyl-ribofuranosyl in the treatment of infections        caused by viruses that replicate through an RNA dependent RNA        polymerase; and    -   (g) use of the 3′-L-valine ester of        β-D-2′-C-methyl-ribofuranosyl in the treatment of viral        infections by administration in combination or alternation with        another antiviral agent.

In an alternative embodiment, the active compound is a 3′-amino acidester of β-D-2′-C-methyl-ribofuranosyl cytidine, wherein the amino acidcan be natural or synthetic and can be in a D or L stereoconfiguration.In another embodiment, the active compound is a 3′-acyl ester of β-D orβ-L 2′-C-methyl-ribofuranosyl cytidine. The compounds of this inventioneither possess antiviral activity, or are metabolized to a compound thatexhibits such activity.

Although not to be bound by theory, in vitro mechanism-of-action studiessuggest that mCyd is a specific inhibitor of genomic RNA replication.Moreover, the intracellular 5′-triphosphate moiety, mCyd-TP, appears todirectly inhibit the NS5B RNA-dependent-RNA polymerase. Analysis of RNAsynthesis in the presence of mCyd-TP suggests that mCyd-TP acts as aspecific chain terminator of viral RNA synthesis through as yetunidentified mechanisms. Antiviral nucleosides and nucleoside analogsare generally converted into the active metabolite, the 5′-triphosphate(TP) derivatives by intracellular kinases. The nucleoside-TPs then exerttheir antiviral effect by inhibiting the viral polymerase during virusreplication. In cultured cells, intracellular phosphorylation convertsmCyd predominantly into the active species, mCyd-triphosphate (mCyd-TP),along with low levels of mCyd-diphosphate. Additionally, a second TPproduct, 2′-C-methyl-uridine TP (mUrd-TP), is found and is thought toarise via intracellular deamination of mCyd or mCyd-5′-phosphatespecies.

Flaviviruses included within the scope of this invention are discussedgenerally in Fields Virology, Editors: Fields, N., Knipe, D. M. andHowley, P. M.; Lippincott-Raven Pulishers, Philadelphia, Pa.; Chapter 31(1996). Specific flaviviruses include, without limitation: Absettarov;Alfiy; Apoi; Aroa; Bagaza; Banzi; Bououi; Bussuquara; Cacipacore; CareyIsland; Dakar bat; Dengue viruses 1, 2, 3 and 4; Edge Hill; Entebbe bat;Gadgets Gully; Hanzalova; Hypr; Ilheus; Israel turkeymeningoencephalitis; Japanese encephalitis; Jugra; Jutiapa; Kadam;Karshi; Kedougou; Kokoera; Koutango; Kumlinge; Kunjin; Kyasanur Forestdisease; Langat; Louping ill; Meaban; Modoc; Montana myotisleukoencephalitis; Murray valley encephalitis; Naranjal; Negishi; Ntaya;Omsk hemorrhagic fever; Phnom-Penh bat; Powassan; Rio Bravo; Rocio;Royal Farm; Russian spring-summer encephalitis; Saboya; St. Louisencephalitis; Sal Vieja; San Perlita; Saumarez Reef; Sepik; Sokuluk;Spondweni; Stratford; Temusu; Tyuleniy; Uganda S, Usutu, Wesselsbron;West Nile; Yaounde; Yellow fever; and Zika.

Pestiviruses included within the scope of this invention are alsodiscussed generally in Fields Virology (Id.). Specific pestivirusesinclude, without limitation: bovine viral diarrhea virus (“VDV”);classical swine fever virus (“CSFV”) also known as hog cholera virus);and border disease virus (“DV”).

DEFINITIONS

The term alkyl, as used herein, unless otherwise specified, refers to asaturated straight, branched, or cyclic, primary, secondary, or tertiaryhydrocarbon of typically C₁ to C₁₀, and specifically includes CF₃, CCl₃,CFCl₂, CF₂Cl, CH₂CF₃, CF₂CF₃, methyl, ethyl, propyl, isopropyl,cyclopropyl, butyl, isobutyl, secbutyl, t-butyl, pentyl, cyclopentyl,isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl,3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl. The termincludes both substituted and unsubstituted alkyl groups, andparticularly includes halogenated alkyl groups, and even moreparticularly fluorinated alkyl groups. Non-limiting examples of moietieswith which the alkyl group can be substituted are selected from thegroup consisting of halogen (fluoro, chloro, bromo or iodo), hydroxyl,amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonicacid, sulfate, phosphonic acid, phosphate, or phosphonate, eitherunprotected, or protected as necessary, as known to those skilled in theart, for example, as taught in Greene, et al., Protective Groups inOrganic Synthesis, John Wiley and Sons, Second Edition, 1991, herebyincorporated by reference.

The term lower alkyl, as used herein, and unless otherwise specified,refers to a C₁ to C₄ saturated straight, branched, or a cyclic (forexample, cyclopropyl)alkyl group, including both substituted andunsubstituted forms. Unless otherwise specifically stated in thisapplication, when alkyl is a suitable moiety, lower alkyl is preferred.Similarly, when alkyl or lower alkyl is a suitable moiety, unsubstitutedalkyl or lower alkyl is preferred.

The term alkylamino or arylamino refers to an amino group that has oneor two alkyl or aryl substituents, respectively.

The term “protected” as used herein and unless otherwise defined refersto a group that is added to an oxygen, nitrogen, or phosphorus atom toprevent its further reaction or for other purposes. A wide variety ofoxygen and nitrogen protecting groups are known to those skilled in theart of organic synthesis.

The term aryl, as used herein, and unless otherwise specified, refers tophenyl, biphenyl, or naphthyl, and preferably phenyl. The term includesboth substituted and unsubstituted moieties. The aryl group can besubstituted with any desired moiety, including, but not limited to, oneor more moieties selected from the group consisting of halogen (fluoro,chloro, bromo or iodo), hydroxyl, amino, alkylamino, arylamino, alkoxy,aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid,phosphate, or phosphonate, either unprotected, or protected asnecessary, as known to those skilled in the art, for example, as taughtin Greene, et al., Protective Groups in Organic Synthesis, John Wileyand Sons, Second Edition, 1991.

The term alkaryl or alkylaryl refers to an alkyl group with an arylsubstituent. The term aralkyl or arylalkyl refers to an aryl group withan alkyl substituent.

The term halo, as used herein, includes a specific description ofchloro, bromo, iodo, and fluoro individually.

The term purine or pyrimidine base includes, but is not limited to,adenine, N⁶-alkylpurines, N⁶-acylpurines (wherein acyl is C(O)(alkyl,aryl, alkylaryl, or arylalkyl), N⁶-benzylpurine, N⁶-halopurine,N⁶-vinylpurine, N⁶-acetylenic purine, N⁶-acyl purine, N⁶-hydroxyalkylpurine, N⁶-alkylaminopurine, N⁶-thioalkyl purine, N²-alkylpurines,N²-alkyl-6-thiopurines, thymine, cytosine, 5-fluorocytosine,5-methylcytosine, 6-azapyrimidine, including 6-azacytosine, 2- and/or4-mercaptopyrmidine, uracil, 5-halouracil, including 5-fluorouracil,C⁵-alkylpyrimidines, 5-iodo-pyrimidine, 6-iodo-pyrimidine,2-Br-vinyl-5-pyrimidine, 2-Br-vinyl-6-pyrimidine, C⁵-benzylpyrimidines,C⁵-halopyrimidines, C⁵-vinylpyrimidine, C⁵-acetylenic pyrimidine,C⁵-acyl pyrimidine, C⁵-hydroxyalkyl purine, C⁵-amidopyrimidine,C⁵-cyanopyrimidine, C⁵-nitropyrimidine, C⁵-amino-pyrimidine,N²-alkylpurines, N²-alkyl-6-thiopurines, 5-azacytidinyl, 5-azauracilyl,triazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, andpyrazolopyrimidinyl. Purine bases include, but are not limited to,guanine, adenine, hypoxanthine, 2,6-diaminopurine, and 6-chloropurine.Functional oxygen and nitrogen groups on the base can be protected asnecessary or desired. Suitable protecting groups are welt known to thoseskilled in the art, and include trimethylsilyl, dimethylhexylsilyl,t-butyldimethylsilyl, and t-butyldiphenylsilyl, trityl, alkyl groups,and acyl groups such as acetyl and propionyl, methanesulfonyl, andp-toluenesulfonyl.

The term acyl or O-linked ester refers to a group of the formula C(O)R′,wherein R′ is an straight, branched, or cyclic alkyl (including loweralkyl), carboxylate residue of an amino acid, aryl including phenyl,heteroaryl, alkaryl, aralkyl including benzyl, alkoxyalkyl includingmethoxymethyl, aryloxyalkyl such as phenoxymethyl; or substituted alkyl(including lower alkyl), aryl including phenyl optionally substitutedwith chloro, bromo, fluoro, iodo, C₁ to C₄ alkyl or C₁ to C₄ alkoxy,sulfonate esters such as alkyl or aralkyl sulphonyl includingmethanesulfonyl, the mono, di or triphosphate ester, trityl ormonomethoxy-trityl, substituted benzyl, alkaryl, aralkyl includingbenzyl, alkoxyalkyl including methoxymethyl, aryloxyalkyl such asphenoxymethyl. Aryl groups in the esters optimally comprise a phenylgroup. In nonlimiting embodiments, acyl groups include acetyl,trifluoroacetyl, methylacetyl, cyclopropylacetyl, cyclopropyl-carboxy,propionyl, butyryl, isobutyryl, hexanoyl, heptanoyloctanoyl,neo-heptanoyl, phenylacetyl, 2-acetoxy-2-phenylacetyl, diphenylacetyl,α-methoxy-α-trifluoromethyl-phenylacetyl, bromoacetyl,2-nitro-benzeneacetyl, 4-chloro-benzeneacetyl,2-chloro-2,2-diphenylacetyl, 2-chloro-2-phenylacetyl, trimethylacetyl,chlorodifluoroacetyl, perfluoroacetyl, fluoroacetyl,bromodifluoroacetyl, methoxyacetyl, 2-thiopheneacetyl,chlorosulfonylacetyl, 3-methoxyphenylacetyl, phenoxyacetyl,tert-butylacetyl, trichloroacetyl, monochloro-acetyl, dichloroacetyl,7H-dodecafluoro-heptanoyl, perfluoro-heptanoyl,7H-dodeca-fluoroheptanoyl, 7-chlorododecafluoro-heptanoyl,7-chloro-dodecafluoro-heptanoyl, 7H-dodecafluoroheptanoyl,7H-dodeca-fluoroheptanoyl, nona-fluoro-3,6-dioxa-heptanoyl,nonafluoro-3,6-dioxaheptanoyl, perfluoroheptanoyl, methoxybenzoyl,methyl 3-amino-5-phenylthiophene-2-carboxyl,3,6-dichloro-2-methoxy-benzoyl, 4-(1,1,2,2-tetrafluoro-ethoxy)-benzoyl,2-bromo-propionyl, omega-aminocapryl, decanoyl, n-pentadecanoyl,stearyl, 3-cyclopentyl-propionyl, 1-benzene-carboxyl, O-acetylmandelyl,pivaloyl acetyl, 1-adamantane-carboxyl, cyclohexane-carboxyl,2,6-pyridinedicarboxyl, cyclopropane-carboxyl, cyclobutane-carboxyl,perfluorocyclohexyl carboxyl, 4-methylbenzoyl, chloromethyl isoxazolylcarbonyl, perfluorocyclohexyl carboxyl, crotonyl,1-methyl-1H-indazole-3-carbonyl, 2-propenyl, isovaleryl,1-pyrrolidinecarbonyl, 4-phenylbenzoyl.

The term amino acid includes naturally occurring and synthetic α, β, γ,or δ amino acids, and includes but is not limited to, amino acids foundin proteins, i.e. glycine, alanine, valine, leucine, isoleucine,methionine, phenylalanine, tryptophan, proline, serine, threonine,cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine,arginine and histidine. In a preferred embodiment, the amino acid is inthe L-configuration, but can also be used in the D-configuration.Alternatively, the amino acid can be a derivative of alanyl, valinyl,leucinyl, isoleuccinyl, prolinyl, phenylalaninyl, tryptophanyl,methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl,asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl, argininyl,histidinyl, β-alanyl, β-valinyl, β-leucinyl, β-isoleuccinyl, β-prolinyl,β-phenylalaninyl, β-tryptophanyl, β-methioninyl, β-glycinyl, β-serinyl,β-threoninyl, β-cysteinyl, β-tyrosinyl, β-asparaginyl, β-glutaminyl,β-aspartoyl, β-glutaroyl, β-lysinyl, β-argininyl or β-histidinyl.

As used herein, the term “substantially free of” or “substantially inthe absence of” refers to a nucleoside composition that includes atleast 85 or 90% by weight, preferably 95%, 98, 99% or 100% by weight, ofthe designated enantiomer of that nucleoside.

Similarly, the term “isolated” refers to a nucleoside composition thatincludes at least 85 or 90% by weight, preferably 95%, 98%, 99% or 100%by weight, of the nucleoside.

The term host, as used herein, refers to an unicellular or multicellularorganism in which the virus can replicate, including cell lines andanimals, and preferably a human. Alternatively, the host can be carryinga part of the Flaviviridae viral genome, whose replication or functioncan be altered by the compounds of the present invention. The term hostspecifically refers to infected cells, cells transfected with all orpart of the Flaviviridae genome and animals, in particular, primates(including chimpanzees) and humans. In most animal applications of thepresent invention, the host is a human patient. Veterinary applications,in certain indications, however, are clearly anticipated by the presentinvention (such as chimpanzees).

The term “pharmaceutically acceptable salt or prodrug” is usedthroughout the specification to describe any pharmaceutically acceptableform (such as an ester, phosphate ester, salt of an ester or a relatedgroup) of a nucleoside compound which, upon administration to a patient,provides the nucleoside compound. Pharmaceutically acceptable saltsinclude those derived from pharmaceutically acceptable inorganic ororganic bases and acids. Suitable salts include those derived fromalkali metals such as potassium and sodium, alkaline earth metals suchas calcium and magnesium, among numerous other acids well known in thepharmaceutical art. Pharmaceutically acceptable prodrugs refer to acompound that is metabolized, for example hydrolyzed or oxidized, in thehost to form the compound of the present invention. Typical examples ofprodrugs include compounds that have biologically labile protectinggroups on a functional moiety of the active compound. Prodrugs includecompounds that can be oxidized, reduced, aminated, deaminated,hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated,dealkylated, acylated, deacylated, phosphorylated, dephosphorylated toproduce the active compound. The compounds of this invention possessantiviral activity against a Flaviviridae, or are metabolized to acompound that exhibits such activity.

I. Active Compounds, Physiologically Acceptable Salts and ProdrugsThereof

Methods and compositions for the treatment of pestivirus, flavivirus andhepatitis C virus infection are described that include administering aneffective amount of 2-C-methyl-cytidine-3′-O-L-valine, or apharmaceutically acceptable salt, ester or prodrug thereof to a host inneed thereof.

In one embodiment, a hydrochloride salt of the compound of Formula (I)is used. In another embodiment, a dihydrochloride salt of the compoundof Formula (I) is preferred. However, the active compound can beadministered as any salt or prodrug that upon administration to therecipient is capable of providing directly or indirectly the parentcompound, or that exhibits activity itself. Nonlimiting examples are thepharmaceutically acceptable salts, which are alternatively referred toas “physiologically acceptable salts”, and a compound that has beenalkylated, acylated or otherwise modified at the 5′-position or on thepurine or pyrimidine base, thereby forming a type of “pharmaceuticallyacceptable prodrug”. Further, the modifications can affect thebiological activity of the compound, in some cases increasing theactivity over the parent compound. This can easily be assessed bypreparing the salt or prodrug and testing its antiviral activityaccording to the methods described herein, or other methods known tothose skilled in the art.

In a first principal embodiment a compound of the Formula (I), or apharmaceutically acceptable salt or prodrug thereof, is provided:

It is to be understood that all stereoisomeric, tautomeric andpolymorphic forms of the compounds are included herein. The 2′-methylsubstitution also may be other alkyl groups such as ethyl, propyl, or,alternatively, ethenyl.

In an alternative embodiment, the active compound is a 3′-amino acidester of β-D-2′-C-methyl-ribofuranosyl cytidine, wherein the amino acidcan be natural or synthetic and can be in a D or L stereoconfiguration.In another embodiment, the active compound is a 3′-acyl ester ofβ-D-2′-C-methyl-ribofuranosyl cytidine.

In another alternative embodiment, the 5′-hydroxyl group is replacedwith a 5′-OR, wherein R is phosphate (including monophosphate,diphosphate, triphosphate, or a stabilized phosphate prodrug); astabilized phosphate prodrugi acyl (including lower acyl); alkyl(including lower alkyl); sulfonate ester including alkyl or arylalkylsulfonyl including methanesulfonyl and benzyl, wherein the phenyl groupis optionally substituted with one or more substituents as described inthe definition of aryl given herein; a lipid, including a phospholipids;an amino acid; a carbohydrate; a peptide; cholesterol; or otherpharmaceutically acceptable leaving group which when administered invivo is capable of providing a compound wherein R is independently H orphosphate.

II. Synthesis of Active Compounds

The compound of the present invention can be synthesized by means knownin the art. For example, in one embodiment, a nucleoside, nucleosideanalog, or a salt, prodrug, stereoisomer or tautomer thereof, that isdisubstituted at the 2° C. is prepared. In another embodiment,β-D-2′-C-methyl-cytidine(4-amino-1-(3,4-dihydroxy-5-hydroxymethyl-3-C-methyl-tetrahydro-furan-2-yl)-1H-pyrimidine-2-one),which has value as an active nucleoside or is used as a processintermediate is prepared. In yet another embodiment, 3′-O-valinyl esterof β-D-2′-C-methyl-cytidine (2-amino-3-methyl-butyric acid5-(4-amino-2-oxo-2H-pyrimidin-1-yl)-4-hydroxy-4-C-methyl-2-hydroxymethyl-tetrahydro-furan-3-ylester) or a hydrochloride salt form is prepared. Nucleosides, nucleosideanalogs, salts or ester prodrugs prepared may be used as intermediatesin the preparation of a wide variety of other nucleoside analogues, ormay be used directly as antiviral and/or antineoplastic agents.

EXAMPLE 1 Synthesis of β-D-2′-C-methyl-ribofuranosylcytidine-3′-O-L-valine ester

In one synthesis method, depicted in FIG. 1 a, the synthesis comprisesreacting cytosine, BSA and SnCl₄/acetonitrile with1,2,3,5-tetra-O-benzoyl-2-C-methyl-β-D-ribofuranose (1) to form4-amino-1-(3,4-dibenzoyloxy-5-benzoyloxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidin-2-one(2; and reacting (2) with NaOMe/MeOH to provide 4-amino-1(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidin-2-one(3), also known as 2-C-methyl-β-D-ribofuranose. The use of cytosine as astarting material rather than benzoyl-cytosine improves the “atomeconomy” of the process and simplifies purification at later steps.

The next steps in this process comprise reacting (3) with Me₂NCH(OMe)₂in DMF to form (4),N-[1-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-2-oxo-1,2-dihydro-pyrimidin-4-yl]-N,N-dimethyl-formamidine,which is the amino-protected form of (3); reacting (4) with TBDPSCl andimidazole in DCM to provide the 5′-silyl-protected form of (4) asN′-{1-[5-(tert-butyl-diphenyl-silanyloxymethyl)-3,4-dihydroxy-3-methyl-tetrahydro-furan-2-yl]-2-oxo-1,2-dihydro-pyrimidin-4-yl}-N,N-dimethyl-formamidine(5), where the use of DCM provides the advantage of having greatercontrol over disilyl by-product formation; reacting (5) withN-Boc-L-valine, EDC and DMAP in DCM at room temperature to form2-tert-butoxycarbonylamino-3-methyl-butyric acid2-(tert-butyl-diphenyl-silanyloxy-methyl)-5-[4-(dimethylamino-methyleneamino)-2-oxo-2H-pyrimidin-1-yl]-4-hydroxy-4-methyl-tetrahydro-furan-3-ylester (6); removing the silyl and amino-protecting groups by reacting(6) with NH₄F in MeOH in the presence of approximately 10 moleequivalents of ethyl acetate to prevent cleavage of the 3′-O-valinylester by liberated ammonia, and refluxing the mixture to provide2-tert-butoxycarbonylamino-3-methyl-butyric acid5-(4-amino-2-oxo-2H-pyrimidin-1-yl)-4-hydroxy-2-hydroxymethyl-4-methyl-tetrahydro-furan-3-ylester to provide (7); and finally, reacting (7) with HCl in EtOH toprovide 2-amino-3-methyl-butyric acid5-(4-amino-2-oxo-2H-pyrimidin-1-yl)-4-hydroxy-2-hydroxymethyl-4-methyl-tetrahydro-furan-3-ylester, dihydrochloride salt (8) as a final product.

Alternative Synthesis

In another method to synthesize the compounds of the invention, shown inFIG. 1 b, benzoylcytosine, BSA and SnCl₄/acetonitrile are reacted with1,2,3,5-tetra-O-benzoyl-2-C-methyl-β-D-ribofuranose (1) to form4-benzoylamino-1-(3,4-dibenzoyloxy-5-benzoyloxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidin-2-one(2a); reacting (2a) with NH₃ in methanol and chromatographicallyseparating the product,4-amino-1-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidin-2-one(3), also known as β-D-2′-C-methyl-cytidine; reacting (3) withMe₂NCH(OMe)₂ in DMF at room temperature for 1.5 hours to formN-[1-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-2-oxo-1,2-dihydro-pyrimidin-4-yl]-N,N-dimethyl-formamidine(4); reacting (4) with TBDPSCl and pyridine at room temperature for 6hours to provideN′-{1-[5-(tert-butyl-diphenyl-silanyloxymethyl)-3,4-dihydroxy-3-methyl-tetrahydro-furan-2-yl]-2-oxo-1,2-dihydro-pyrimidin-4-yl}-N,N-dimethyl-formamidine(5); reacting (5) with N-Boc-L-valine, DEC and DMAP in THF/DMF at roomtemperature for 2 days and subjecting the product formed from thisreaction to HPLC in order to provide2-tert-butoxycarbonylamino-3-methyl-butyric acid2-(tert-butyl-diphenyl-silanyloxy-methyl)-5-[4-dimethylaminomethyleneamino)-2-oxo-2H-pyrimidin-1-yl]-4-hydroxy-4-methyl-tetrahydro-furan-3-ylester (6); refluxing (6) with NH₄F in MeOH for about 3 hours to removethe silyl and amino-protecting groups, and subjecting the product tochromatographic purification to provide2-tert-butoxycarbonylamino-3-methyl-butyric acid5-(4-amino-2-oxo-2H-pyrimidin-1-yl)-4-hydroxy-2-hydroxymethyl-4-methyl-tetrahydro-furan-3-ylester (7); and finally reacting (7) with HCl in EtOAc at roomtemperature to provide 2-amino-3-methyl-butyric acid5-(4-amino-2-oxo-2H-pyrimidin-1-yl)-4-hydroxy-2-hydroxymethyl-4-methyl-tetrahydro-furan-3-ylester, dihydrochloride salt (8) as a final product.

EXAMPLE 2 Synthesis of 2′-C-methyl-cytidine-3′-O-L-valine ester(val-mCyd)

Step 1: Synthesis of Compound 9 2-C-Methyl-D-ribonic-γ-lactone

De-ionized water (100 mL) was stirred in a 250 mL 3-necked round bottomflask, equipped with an overhead stirrer, a stirring shaft, a digitaltemperature read-out device and an argon line. Argon was bubbled intowater for thirty minutes and D-fructose (20.0 g, 0.111 mole) was addedand the solution became clear in a few minutes. Calcium oxide (12.5 g,0.223 mole) was added in portions over a period of five minutes and themixture was vigorously stirred. An exotherm was observed and reactiontemperature reached 39.6° C. after 10 minutes from the start of thecalcium oxide addition. After about fifteen minutes, the reactionmixture developed a yellow color that deepened with time. After threehours, an aliquot was withdrawn for TLC analysis. The aliquot wasacidified to pH 2 using saturated aqueous solution of oxalic acid. Theresulting white suspension was evaporated under reduced pressure toremove the water. Toluene (2 mL) was added to the residue and themixture was evaporated under reduced pressure (at 45-50° C.) to removeany trace of water. The residual solid was reconstituted in 2 mL of 1:1tetrahydrofluran:methanol mixture. After thorough mixing, the suspensionwas allowed to settle and the supernatant clear solution was spotted forTLC (silica plate was developed in 2% methanol in ethyl acetate andstained in 1% alkaline potassium permanganate dip. The plate was thenheated, using a heat gun, until the appearance of yellowish spots on thepink background). The desired lactone typically appears at an R_(f)value of 0.33 under the above conditions. More polar by-products andunreacted material are detected in the R_(f) value range of 0.0 to 0.2.

Although product formation was observed after 3 hours, the reaction wasallowed to continue for 22 hours during which time the reaction mixturewas stirred at 25° C. At the end of this period, pH of the mixture was13.06. Carbon dioxide gas was bubbled into the reaction mixture forabout 2.5 hours (pH was 7.25). The formed calcium carbonate solid wasremoved by vacuum filtration, filter cake washed with 50 mL ofde-ionized water. The aqueous layers were combined and treated withoxalic acid (5.0 g, 0.056 mole) and the mixture was vigorously stirredat 25° C. for 30 minutes (The initial dark color largely disappeared andthe mixture turned into a milky white slurry). The pH of the mixture atthis stage is typically 2-3. The slurry mixture was stirred at 45-50° C.overnight. The mixture was then evaporated under reduced pressure and at45-50° C. to remove 75 mL of water. Sodium chloride (30 g) andtetrahydrofuran (100 mL) were added to the aqueous slurry (about 75 mL)and the mixture was vigorously stirred at 25° C. for 30 minutes. Thelayers were separated and the aqueous layer was stirred for 10 minuteswith 75 mL of fresh tetrahydrofuran. This process was repeated for threetimes and the tetrahydrofuran solutions were combined and stirred with10 g of anhydrous magnesium sulfate for 30 minutes. The mixture wasfiltered and the magnesium sulfate filter cake was washed with 60 mL oftetrahydrofuran. The filtrate was evaporated under reduced pressure andat 40° C. to give 10.86 g of crude product as a dark orange semisolid.(For scale up runs tetrahydrofuran will be replaced with acetone insteadof evaporation of crude product to dryness). Crude product was stirredwith acetone (20 mL) at 20° C. for 3 hours. Product was collected byvacuum filtration and the filter cake washed with 12 mL of acetone togive the desired product 9 as white crystalline solid. Product was driedin vacuum to give 2.45 g (13.6% yield). Melting point of compound 9:158-162° C. (literature melting point: 160-161° C.). ¹H NMR (DMSO-d₆) δppm 5.69 (s, 1H, exch. With D₂O), 5.41 (d, 1H, exch. With D₂O), 5.00 (t,1H, exch. With D₂O), 4.15 (m, 1H), 3.73 (m, 2H), 3.52 (m, 1H), 1.22 (s,3H). ¹³C NMR (DMSO-d₆) δ ppm 176.44, 82.95, 72.17, 72.02, 59.63, 20.95.(C₆H₁₀O₅: calcd C, 44.45; H, 6.22. Found: C, 44.34; H, 6.30).

Step 2: Synthesis of Compound 102,3,5-Tri-O-benzoyl-2-C-methyl-D-ribonic-γ-lactone

A mixture of lactone 1 (3.0 g, 18.50 mmol.), 4-dimethylaminopyridine(0.45 g, 3.72 mmol.) and triethylamine (25.27 g, 249.72 mmol.) in1,2-dimethoxy ethane (50 mL) was stirred at 25° C. under argonatmosphere for thirty minutes. This white suspension was cooled to 5° C.and benzoyl chloride (11.7 g, 83.23 mmol.) was added over a period offifteen minutes. The mixture was stirred at 25° C. for two hours. TLCanalysis (silica, 2% methanol in ethyl acetate) indicated completeconsumption of starting material. Ice cold water (100 g) was added tothe reaction mixture and stirring was continued for thirty minutes. Theformed white solids were collected by vacuum filtration and filter cakewashed with cold water (50 mL). This crude product was stirred withtert-butyl methyl ether (60 mL) at 20° C. for thirty minutes, thenfiltered, filter cake washed with tert-butyl methyl ether (25 mL) anddried in vacuum to give 7.33 g (83.4% yield) of compound 10 as a whitesolid in 97.74% purity (HPLC/AUC). Melting point of compound 10:137-140° C. (literature melting point: 141-142° C.). ¹H NMR (CDCl₃) δppm 8.04 (d, 2H), 7.92 (d, 2H), 7.73 (d, 2H), 7.59 (t, 1H), 7.45 (m,4H), 7.32 (t, 2H), 7.17 (t, 2H), 5.51 (d, 1H), 5.17 (m, 1H), 4.82-4.66(d of an AB quartet, 2H) 1.95, (s, 3H). ¹³C NMR (CDCl₃) δ ppm 172.87,166.17, 166.08, 165.58, 134.06, 133.91, 133.72, 130.09, 129.85, 129.80,129.37, 128.78, 128.60, 128.49, 127.96, 127.89, 79.67, 75.49, 72.60,63.29, 23.80. TOF MS ES+ (M+1: 475).

Step 3: Synthesis of Compound 112,3,5-Tri-O-benzoyl-2-C-methyl-β-D-ribofuranose

A solution of Red-Al (65 wt. % in toluene, 2.0 mL, 6.56 mmol.) inanhydrous toluene (2.0 mL) was stirred at 0° C. under argon atmosphere.A solution of anhydrous ethanol (0.38 mL, 6.56 mmol.) in anhydroustoluene (1.6 mL) was added to the toluene solution over a period of fiveminutes. The resulting mixture was stirred at 0° C. for fifteen minutesand 2 mL (2.18 mmol.) of this Red-Al/ethanol reagent was added to a cold(−5° C.) solution of 2,3,5-tri-O-benzoyl-2-C-methyl-D-ribonolactone 2(475 mg, 1.0 mmol.) in anhydrous toluene (10 mL) over a period of 10minutes. The reaction mixture was stirred at −5° C. for forty minutes.TLC analysis (silica plates, 35% ethyl acetate in heptane) indicatedcomplete consumption of starting material. HPLC analysis indicated only0.1% of starting material remaining. The reaction was quenched withacetone (0.2 mL), water (15 mL) and 1 N HCl (15 mL) at 0° C. and allowedto warm to room temperature. 1 N HCl (5 mL) was added to dissolve theinorganic salts (pH: 2-3). The mixture was extracted with ethyl acetate(3×25 mL) and the organic solution washed with brine (25 mL), dried(anhydrous sodium sulfate, 10 g) and solvent removed under reducedpressure and at temperature of 40° C. to give the desired product 11 inquantitative yield (480 mg). This material was used as is for thesubsequent step.

Step 4: Synthesis of Compound 121,2,3,5-tetra-O-benzoyl-2-C-methyl-β-D-ribofuranose

Benzoyl chloride (283 mg, 2.0 mmol.) was added, over a period of fiveminutes, to a cold solution (5° C.) of compound II (480 mg, 1.0 mmol.),4-dimethylaminopyridine (12.3 mg, 0.1 mmol.) and triethylamine (506 mg,5.0 mmol.) in anhydrous tetrahydrofuran (5 mL). The reaction mixture wasstirred at room temperature and under argon atmosphere overnight. HPLCanalysis indicated 0.25% of un-reacted starting material. The reactionwas quenched by adding ice-cold water (10 g) and saturated aqueoussolution of sodium bicarbonate. Tetrahydrofuran was removed underreduced pressure and the mixture was extracted with ethyl acetate (50mL). The organic solution was washed with water (25 mL), brine (25 mL),dried (anhydrous sodium sulfate, 12 g) and solvent removed under reducedpressure to give 650 mg of thick oily product. This crude product wasstirred with 5 mL of tert-butyl methyl ether for 5 minutes and heptane(5 mL) and water (0.1 mL) were added and stirring was continued for anadditional period of two hours at 20° C. Solids were collected by vacuumfiltration and filter caked washed with 1:1 heptane:tert-butyl methylether solution (6 mL) and tert-butyl methyl ether (2 mL). Drying thesolid in vacuum gave 300 mg (52%) of desired product 12 (98.43% pure byHPLC/AUC) as a white solid that melted at 154-156.3° C. (literaturemelting point: 155-156° C.). ¹H NMR (CDCl₃) δ ppm 8.13 (m, 4H), 8.07 (d,2H), 7.89 (d, 2H), 7.63 (m, 3H), 7.48 (m, 6H), 7.15 (m, 3H), 7.06 (s,1H), 5.86 (dd, 1H), 4.79 (m, 1H), 4.70-4.52 (d of an AB quartet, 2H),1.95, (s, 3H). ¹³C NMR (CDCl₃) δ ppm 166.31, 165.83, 165.01, 164.77,134.01, 133.86, 133.70, 133.17, 130.44, 130.13, 129.97, 129.81, 129.59,129.39, 129.07, 128.84, 128.76, 128.37, 98.01, 86.87, 78.77, 76.35,64.05, 17.07. (C₃₄H₂₈O₉: calcd C, 70.34; H, 4.86. Found: C, 70.20; H,4.95).

Step 5: Synthesis of Compound 134-Amino-1-(3,4-dibenzoyloxy-5-benzyloxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidine-2-one

Cytosine (89 g, 0.80 mol) was suspended in acetonitrile (900 ml) in a 12L round bottomed flask equipped with a reflux condenser, overheadstirrer and an argon inlet adapter. The suspension was stirred at 20° C.under argon atmosphere and N,O-bis(trimethylsilyl)acetamide (537 ml, 2.2mol) was added in one portion. The resulting solution was heated to 80°C. and stirred for an additional hour at the same temperature.1,2,3,5-tetra-O-benzoyl-2-C-methyl-β-D-ribofuranose (425.0 g, 0.73 mol)was suspended in acetonitrile (4000 ml) and added to the reactionmixture. The reaction mixture became clear after a few minutes and thetemperature dropped to ca. 50° C. Tin(IV) chloride (154 ml, 1.31 mol)was added over a period of 15 minutes and stirring was continued at 80°.After one hour, an aliquot of reaction mixture was quenched by addingaqueous sodium bicarbonate solution and extracting the aqueous layerwith ethyl acetate. The ethyl acetate layer was examined by TLC (silicagel, 20% ethyl acetate in heptane, R_(f) for sugar derivative: 0.40).TLC analysis indicated the complete consumption of the sugar derivative.Desired product was detected by TLC using 10% methanol indichloromethane (R_(f): 0.37). The reaction was also monitored by HPLC(Method # 2). Reaction mixture was cooled to 20° C. and quenched byadding saturated aqueous sodium bicarbonate solution (3000 ml) over aperiod of 30 minutes (observed an exotherm when added the first fewdrops of the sodium bicarbonate solution). Solid sodium bicarbonate(1350 g) was added in portions to avoid foaming. The mixture was checkedto make sure that its pH is ≧7. Agitation was stopped and layers wereallowed to separate for 20 minutes. The aqueous layer was drained andstirred with ethyl acetate (1500 ml) and the mixture was allowed toseparate (30 minutes). The organic layer was isolated and combined withthe acetonitrile solution. The organic solution was washed with brine(500 ml) and then solvent stripped to a volume of ca. 750 ml. Productcan be used as is in the subsequent reaction. It may also be furtherstripped to white foamy solid, in quantitative yield. Structure ofcompound 10 was confirmed by ¹H NMR analysis.

Step 6: Synthesis of Compound mCyd4-Amino-1-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidine-2-one

Sodium methoxide (13.8 g, 0.26 mol) was added to a solution of compound10 (416 g, 0.73 mol) in methanol (2000 ml). The reaction mixture wasstirred at room temperature and monitored by TLC (silica gel, 10%methanol in dichloromethane, R_(f) of compound 9: 0.53) and (silica gel,30% methanol in dichloromethane, R_(f) of compound 11: 0.21). Productstarted to precipitate after 30 minutes and TLC indicated reactioncompletion after two hours. The reaction was also monitored by HPLC(Method # 2). Methanol was removed under reduced pressure to a volume ofca. 500 ml chased with ethanol (2×500 ml) to a volume of ca. 500 ml. Theresidual thick slurry was diluted with 750 ml of ethanol and the mixturewas stirred at 20° C. for one hour. Product was collected by filtration,filter cake washed with ethanol (100 ml) and tert-butyl-methyl ether(100 ml) and dried to give 168 g (90% yield for the two steps) ofproduct 11 in purity of >97% (HPLC/AUC). Product was also analyzed by ¹Hand ¹³C NMR.

Step 7: Synthesis of Compound 142-Tert-butoxycarbonylamino-3-methyl-butyric acid5-(4-amino-2-oxo-2H-pyrimidin-1-yl-4-hydroxy-2-hydroxymethyl-4-methyl-tetrahydro-furan-3-ylester

A solution of N-(tert-butoxycarbonyl)-L-valine (46.50 g, 214 mmol.),carbonyldiimidazole (34.70 g, 214 mmol.), and anhydrous tetrahydrofuran(1000 mL) in a 2 L round bottom flask, was stirred at 25° C. under argonfor 1.5 hours and then at 40-50° C. for 20 minutes. In a separate 5 L5-necked round bottom flask, equipped with an overhead stirrer, coolingtower, temperature probe, addition funnel, and an argon line was added4-amino-1-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidine-2-one(50.0 g, 195 mmol.) and anhydrous N,N-dimethylformamide (1000 mL). Thismixture was heated at 100° C. for 20 minutes until all of thepyrimidine-2-one derivative compound went into solution, and thentriethyl amine (500 mL) and 4-dimethylaminopyridine (2.38 g, 19 mmol)were added to the solution. The mixture was next heated at 97° C. for 20minutes and the tetrahydrofuran solution was added slowly through anaddition funnel over a period of 2 hours, maintaining the temperature nolower than 82° C. The reaction mixture was heated at 82° C. for 1 hourand monitored by HPLC (product=68%, SM=11%, and impurity at about 12min=17%, excluding dimethylaminopyridine). The reaction mixture wascooled to room temperature and then triethylamine and tetrahydrofuranwere removed under vacuum at 30° C. The solution was then neutralizedwith acetic acid to a pH of 7.69. N,N-dimethylformamidine was removedunder vacuum at 35° C. and chased with ethyl acetate (2×200 mL). Thecrude product was stirred with ethyl acetate (500 mL) and water (300mL). The two layers were separated and the aqueous layer was extractedwith ethyl acetate (500 mL). The combined organic layers were washedwith an aqueous saturated brine solution (500 mL). Next the organiclayer was extracted with an aqueous solution of malonic acid (4×400 mL,10 wt. %). The organic layer was checked by TLC (silica, 20% methanol indichloromethane) to make sure that all the desired product was removedfrom the organic layer. The acidic aqueous extracts were combined andcooled in an ice bath and neutralized with triethylamine to a pH of 7.40so that the solids fell out of solution. Ethyl acetate then was added tothe aqueous layer. The white solids were collected by vacuum filtration.Drying the obtained solids in vacuum gave 81.08 g of 99.01 pure (HPLC)first crop.

Step 8: Synthesis of val-mCyd-2-Amino-3-methyl-butyric acid5-(4-amino-2-oxo-2H-pyrimidine-1-yl)-4-hydroxy-2hydroxy-methyl-4-methyl-tetrahydro-furan-3-yl ester (dihydrochloridesalt)

A solution of compound 14 (21.0 g, 0.046 mol) in ethanol (168 ml) wasstirred in a round bottomed flask equipped with an overhead stirrer,temperature probe, argon line and hydrogen chloride gas bubbler.Hydrogen chloride gas (22 g) was bubbled into the clear solution over aperiod of one hour. The reaction temperature was kept below 30° C. usingan ice-water bath. Solid formation started after a few minutes ofintroducing the hydrogen chloride gas. After 4 hours, HPLC (method # 3)showed only 0.8% of starting material. Solids were collected byfiltration and filter cake washed with ethanol (20 ml) and di-ethylether (100 ml). After drying product under vacuum for 16 hours, 19.06 g(96.5%) of val-mCyd was obtained in 97.26% purity (HPLC, method # 3);m.p. 210° C. (brown), 248-250° C. (melted); ¹H NMR (DMSO-d₆) δ ppm 10.0(s, 1H, ½NH₂, D₂O exchangeable), 8.9-8.6 (2 br s, 4H, ½NH₂, NH₃, D₂Oexchangeable), 8.42 (d, 1H, H-6, J₅₋₆=7.9 Hz), 6.24 (d, 1H, H-5,J₅₋₆=7.9 Hz), 5.84 (s, 1H, H-1′), 5.12 (d, 1H, H-3′, J_(3′-4′)=8.8 Hz),4.22 (d, 1H, H-4, J_(3′-4′)=8.7 Hz), 4.0-3.9 (m, 1H, CH), 3.8-3.5 (m,2H, H-5′, H-5″), 2.3-2.1 (m, 1H, CH), 1.16 (s, 3H, CH₃), 1.0 (m, 6H,(CH₃)₂CH); FAB>0 (GT) 713 (2M+H)⁺, 449 (M+G+H)⁺, 357 (M+H)⁺, 246 (S)⁺,112 (B+2H)⁺; FAB<0 (GT) 747 (2M+Cl)⁻, 483 (M+G+Cl)⁻, 391 (M+Cl)⁻, 355(M−H)⁻, 116 (Val)⁻, 110 (B)⁻, 35 (Cl).

Two different HPLC methods were used to analyze the above compounds.Both methods use the following reverse phase column:

Method 1

254 nm. 1.00 ml/min flow rate of an acetonitrile/water linear gradientas described below. 20 minute run time. Five-minute equilibrationbetween runs.

TABLE 1 Retention time of key intermediates: Compound Retention TimeCompound 10 10.2 min Compound 11  9.4 min Compound 12 12.9 min

Method 2

Identification is determined at 272 nm. The column used is a WatersNovapak® C18, 3.9×150 mm ID, 4 μm particle size, 60 Å pore size orequivalent. The chromatographic conditions are as follows: injectionvolume=10 μl, column temperature=25° C., flow rate=1.00 ml/min,ultraviolet detector at 272 nm, run time is 35 minutes. The systemsuitability requirement for the percent relative standard deviation forthe reference standard is not more than 1.0%.

TABLE 2a Purity and impurities are determined at 272 nm: Solvent A - 20nM Solvent B - triethylammonium Acetonitrile, Time (minutes) acetatebuffer HPLC grade. 0.00 100.0 0.0 10.00 85.0 15.0 25.00 5.0 95.0 35.05.0 95.0

TABLE 2b Retention times of key intermediates and final drug substance:Compound Retention Time (minutes) Compound mCyd 2.7-2.8 Compound 14 15.5val-mCyd  9.1Stereochemistry

It is appreciated that nucleosides of the present invention have severalchiral centers and may exist in and be isolated in optically active andracemic forms. Some compounds may exhibit polymorphism. It is to beunderstood that the present invention encompasses any racemic,optically-active, diastereomeric, polymorphic, or stereoisomeric form,or mixtures thereof, of a compound of the invention, which possess theuseful properties described herein. It being well known in the art howto prepare optically active forms (for example, by resolution of theracemic form by recrystallization techniques, by synthesis fromoptically-active starting materials, by chiral synthesis, or bychromatographic separation using a chiral stationary phase).

In particular, since the 1′ and 4′ carbons of the nucleoside are chiral,their nonhydrogen substituents (the base and the CHOR groups,respectively) can be either cis (on the same side) or trans (on oppositesides) with respect to the sugar ring system. The four optical isomerstherefore are represented by the following configurations (whenorienting the sugar moiety in a horizontal plane such that the oxygenatom is in the back): cis (with both groups “up”, which corresponds tothe configuration of naturally occurring β-D nucleosides), cis (withboth groups “down”, which is a normaturally occurring β-Lconfiguration), trans (with the C2′ substituent “up” and the C4′substituent “down”), and trans (with the C2′ substituent “down” and theC4′ substituent “up”). The “D-nucleosides” are cis nucleosides in anatural configuration and the “L-nucleosides” are cis nucleosides in thenormaturally occurring configuration.

Likewise, most amino acids are chiral (designated as L or D, wherein theL enantiomer is the naturally occurring configuration) and can exist asseparate enantiomers.

Examples of methods to obtain optically active materials are known inthe art, and include at least the following.

-   -   i) physical separation of crystals—a technique whereby        macroscopic crystals of the individual enantiomers are manually        separated. This technique can be used if crystals of the        separate enantiomers exist, i.e., the material is a        conglomerate, and the crystals are visually distinct;    -   ii) simultaneous crystallization—a technique whereby the        individual enantiomers are separately crystallized from a        solution of the racemate, possible only if the latter is a        conglomerate in the solid state;    -   iii) enzymatic resolutions—a technique whereby partial or        complete separation of a racemate by virtue of differing rates        of reaction for the enantiomers with an enzyme exists;    -   iv) enzymatic asymmetric synthesis—a synthetic technique whereby        at least one step of the synthesis uses an enzymatic reaction to        obtain an enantiomerically pure or enriched synthetic precursor        of the desired enantiomer;    -   v) chemical asymmetric synthesis—a synthetic technique whereby        the desired enantiomer is synthesized from an achiral precursor        under conditions that produce asymmetry (i.e., chirality) in the        product, which may be achieved using chiral catalysts or chiral        auxiliaries;    -   vi) diastereomer separations—a technique whereby a racemic        compound is reacted with an enantiomerically pure reagent (the        chiral auxiliary) that converts the individual enantiomers to        diastereomers. The resulting diastereomers are then separated by        chromatography or crystallization by virtue of their now more        distinct structural differences and the chiral auxiliary later        removed to obtain the desired enantiomer;    -   vii) first- and second-order asymmetric transformations—a        technique whereby diastereomers from the racemate equilibrate to        yield a preponderance in solution of the diastereomer from the        desired enantiomer or where preferential crystallization of the        diastereomer from the desired enantiomer perturbs the        equilibrium such that eventually in principle all the material        is converted to the crystalline diastereomer from the desired        enantiomer. The desired enantiomer is then released from the        diastereomer;    -   viii) kinetic resolutions—this technique refers to the        achievement of partial or complete resolution of a racemate (or        of a further resolution of a partially resolved compound) by        virtue of unequal reaction rates of the enantiomers with a        chiral, non-racemic reagent or catalyst under kinetic        conditions;    -   ix) enantiospecific synthesis from non-racemic precursors—a        synthetic technique whereby the desired enantiomer is obtained        from non-chiral starting materials and where the stereochemical        integrity is not or is only minimally compromised over the        course of the synthesis;    -   x) chiral liquid chromatography—a technique whereby the        enantiomers of a racemate are separated in a liquid mobile phase        by virtue of their differing interactions with a stationary        phase. The stationary phase can be made of chiral material or        the mobile phase can contain an additional chiral material to        provoke the differing interactions;    -   xi) chiral gas chromatography—a technique whereby the racemate        is volatilized and enantiomers are separated by virtue of their        differing interactions in the gaseous mobile phase with a column        containing a fixed non-racemic chiral adsorbent phase;    -   xii) extraction with chiral solvents—a technique whereby the        enantiomers are separated by virtue of preferential dissolution        of one enantiomer into a particular chiral solvent;    -   xiii) transport across chiral membranes—a technique whereby a        racemate is placed in contact with a thin membrane barrier. The        barrier typically separates two miscible fluids, one containing        the racemate, and a driving force such as concentration or        pressure differential causes preferential transport across the        membrane barrier. Separation occurs as a result of the        non-racemic chiral nature of the membrane which allows only one        enantiomer of the racemate to pass through.        III. Pharmaceutical Compositions

Hosts, including humans, infected with pestivirus, flavivirus, HCV oranother organism replicating through a RNA-dependent RNA viralpolymerase, or for treating any other disorder described herein, can betreated by administering to the patient an effective amount of theactive compound or a pharmaceutically acceptable prodrug or salt thereofin the presence of a pharmaceutically acceptable carrier or dilutent.The active materials can be administered by any appropriate route, forexample, orally, parenterally, intravenously, intradermally,subcutaneously, or topically, in liquid or solid form.

A preferred dose of the compound for pestivirus, flavivirus or HCV willbe in the range from about 1 to 50 mg/kg, preferably 1 to 20 mg/kg, ofbody weight per day, more generally 0.1 to about 100 mg per kilogrambody weight of the recipient per day. Lower doses may be preferable, forexample doses of 0.5-100 mg, 0.5-50 mg, 0.5-10 mg, or 0.5-5 mg perkilogram body weight per day. Even lower doses may be useful, and thusranges can include from 0.1-0.5 mg per kilogram body weight per day. Theeffective dosage range of the pharmaceutically acceptable salts andprodrugs can be calculated based on the weight of the parent nucleosideto be delivered. If the salt or prodrug exhibits activity in itself, theeffective dosage can be estimated as above using the weight of the saltor prodrug, or by other means known to those skilled in the art.

The compound is conveniently administered in unit any suitable dosageform, including but not limited to one containing 7 to 3000 mg,preferably 70 to 1400 mg of active ingredient per unit dosage form. Anoral dosage of 50-1000 mg is usually convenient, including in one ormultiple dosage forms of 50, 100, 200, 250, 300, 400, 500, 600, 700,800, 900 or 1000 mgs. Lower doses may be preferable, for example from10-100 or 1-50 mg. Also contemplated are doses of 0.1-50 mg, or 0.1-20mg or 0.1-10.0 mg. Furthermore, lower doses may be utilized in the caseof administration by a non-oral route, as, for example, by injection orinhalation.

Ideally the active ingredient should be administered to achieve peakplasma concentrations of the active compound of from about 0.2 to 70 μM,preferably about 1.0 to 10 μM. This may be achieved, for example, by theintravenous injection of a 0.1 to 5% solution of the active ingredient,optionally in saline, or administered as a bolus of the activeingredient.

The concentration of active compound in the drug composition will dependon absorption, inactivation and excretion rates of the drug as well asother factors known to those of skill in the art. It is to be noted thatdosage values will also vary with the severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat the concentration ranges set forth herein are exemplary only andare not intended to limit the scope or practice of the claimedcomposition. The active ingredient may be administered at once, or maybe divided into a number of smaller doses to be administered at varyingintervals of time, with or without other anti-viral agents.

A preferred mode of administration of the active compound is oral. Oralcompositions will generally include an inert diluent or an ediblecarrier. They may be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition.

The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a bindersuch as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a disintegrating agent such asalginic acid, Primogel, or corn starch; a lubricant such as magnesiumstearate or Sterotes; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring. When the dosageunit form is a capsule, it can contain, in addition to material of theabove type, a liquid carrier such as a fatty oil. In addition, dosageunit forms can contain various other materials which modify the physicalform of the dosage unit, for example, coatings of sugar, shellac, orother enteric agents.

The compound can be administered as a component of an elixir,suspension, syrup, wafer, chewing gum or the like. A syrup may contain,in addition to the active compounds, sucrose as a sweetening agent andcertain preservatives, dyes and colorings and flavors.

The compound or a pharmaceutically acceptable prodrug or salt thereofcan also be mixed with other active materials that do not impair thedesired action, or with materials that supplement the desired action,such as antibiotics, antifungals, anti-inflammatories, or otherantivirals, including other nucleoside compounds. Solutions orsuspensions used for parenteral, intradermal, subcutaneous, or topicalapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The parental preparation can be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic.

If administered intravenously, preferred carriers are physiologicalsaline or phosphate buffered saline (PBS).

In a preferred embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems, biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation.

Liposomal suspensions (including liposome's targeted to infected cellswith monoclonal antibodies to viral antigens) are also preferred aspharmaceutically acceptable carriers. These may be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811 (which is incorporated herein by reference inits entirety). For example, liposome formulations may be prepared bydissolving appropriate lipid(s), such as sterol phosphatidylethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidylcholine, and cholesterol, in an inorganic solvent that is thenevaporated, leaving behind a thin film of dried lipid on the surface ofthe container. An aqueous solution of the active compound or itsmonophosphate, diphosphate, and/or triphosphate derivatives is thenintroduced into the container. The container is then swirled by hand tofree lipid material from the sides of the container and to disperselipid aggregates, thereby forming the liposomal suspension.

IV. Prodrugs and Derivatives

Salt Formulations

Administration of the nucleoside as a pharmaceutically acceptable saltis within the scope of the invention. Examples of pharmaceuticallyacceptable salts are organic acid addition salts formed with acids,which provide a physiological acceptable anion. These include, forexample, tosylate, methanesulfonate, acetate, citrate, malonate,tartarate, succinate, benzoate, ascorbate, α-ketoglutarate, andα-glycerophosphate, formate, fumarate, propionate, glycolate, lactate,pyruvate, oxalate, maleate, and salicyate. Suitable inorganic salts mayalso be formed, including, sulfate, nitrate, bicarbonate, hydrobromate,carbonate salts, and phosphoric acid. A particularly preferredembodiment is the mono or dihydrochloride salt.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made. In one embodiment, the saltis a hydrochloride salt of the compound. In a further embodiment, thepharmaceutically acceptable salt is a dihydrochloride salt of thecompound of Formula (I). The compounds of this invention possessantiviral activity against flavivirus, pestivirus or HCV, or aremetabolized to a compound that exhibits such activity.

Nucleotide Prodrugs

The nucleosides described herein can be administered as a nucleotideprodrug to increase the activity, bioavailability, stability orotherwise alter the properties of the nucleoside. A number of nucleotideprodrug ligands are known. In general, alkylation, acylation or otherlipophilic modification of the mono-, di- or triphosphate of thenucleoside reduces polarity and allows passage into cells. Examples ofsubstituent groups that can replace one or more hydrogens on thephosphate moiety are alkyl, aryl, steroids, carbohydrates, includingsugars, and alcohols, such as 1,2-diacylglycerol. Many are described inR. Jones and N. Bischoferger, Antiviral Research, 1995, 27:1-17. Any ofthese can be used in combination with the disclosed nucleosides toachieve a desired effect.

The active nucleoside can also be provided as a 5′-phosphoether lipid ora 5′-ether lipid. Non-limiting examples are disclosed in the followingreferences, which are incorporated by reference herein: Kucera, L. S.,N. Iyer, E. Leake, A. Raen, Modest E. K., D. L. W., and C. Piantadosi.1990. “Novel membrane-interactive ether lipid analogs that inhibitinfectious HIV-1 production and induce defective virus formation.” AIDSRes. Hum. Retro Viruses. 6:491-501; Piantadosi, C., J. Marasco C. J., S.L. Morris-Natschke, K. L. Meyer, F. Gumus, J. R. Surles, K. S. Ishaq, L.S. Kucera, N. Iyer, C. A. Wallen, S. Piantadosi, and E. J. Modest. 1991.“Synthesis and evaluation of novel ether lipid nucleoside conjugates foranti-HIV activity.” J. Med. Chem. 34:1408.1414; Hosteller, K. Y., D. D.Richman, D. A. Carson, L. M. Stuhmiller, G. M. T. van Wijk, and H. vanden Bosch., 1992. “Greatly enhanced inhibition of human immunodeficiencyvirus type I replication in CEM and HT4-6C cells by 3′-deoxythymidinediphosphate dimyristoylglycerol, a lipid prodrug of 3-deoxythymidine.”Antimicro. Agents Chemother. 36:2025.2029; Hosetler, K. Y., L. M.Stuhmiller, H. Lenting, H. van den Bosch, and D. D. Richman, 1990.“Synthesis and antiretroviral activity of phospholipid analogs ofazidothymidine and other antiviral nucleosides.” J. Biol. Chem.265:61127.

Nonlimiting examples of U.S. patents that disclose suitable lipophilicsubstituents that can be covalently incorporated into the nucleoside,preferably at the 5′-OH position of the nucleoside or lipophilicpreparations thereof, include U.S. Pat. Nos. 5,149,794 (Sep. 22, 1992,Yatvin et al.); 5,194,654 (Mar. 16, 1993, Hostetler et al., 5,223,263(Jun. 29, 1993, Hostetler et al.); 5,256,641 (Oct. 26, 1993, Yatvin etal.); 5,411,947 (May 2, 1995, Hostetler et al.); 5,463,092 (Oct. 31,1995, Hostetler et al.); 5,543,389 (Aug. 6, 1996, Yatvin et al.);5,543,390 (Aug. 6, 1996, Yatvin et al.); 5,543,391 (Aug. 6, 1996, Yatvinet al.); and 5,554,728 (Sep. 10, 1996; Basava et al.), all of which areincorporated herein by reference. Foreign patent applications thatdisclose lipophilic substituents that can be attached to the nucleosidesof the present invention, or lipophilic preparations, include WO89/02733, WO 90/00555, WO 91/16920, WO 91/18914, WO 93/00910, WO94/26273, WO 96/15132, EP 0 350 287, EP 93917054.4, and WO 91/19721.

V. Combination or Alternation Therapy

The active compounds of the present invention can be administered incombination or alternation with another anti-flavivirus or pestivirusagent, or in particular an anti-HCV agent. In combination therapy,effective dosages of two or more agents are administered together,whereas in alternation or sequential-step therapy, an effective dosageof each agent is administered serially or sequentially. The dosagesgiven will depend on absorption, inactivation and excretion rates of thedrug as well as other factors known to those of skill in the art. It isto be noted that dosage values will also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens and schedules should beadjusted over time according to the individual need and the professionaljudgment of the person administering or supervising the administrationof the compositions. In preferred embodiments, an anti-HCV (oranti-pestivirus or anti-flavivirus) compound that exhibits an EC₅₀ of10-15 μM, or preferably less than 1-5 μM, is desirable.

It has been recognized that drug-resistant variants of flaviviruses,pestiviruses or HCV can emerge after prolonged treatment with anantiviral agent. Drug resistance most typically occurs by mutation of agene that encodes for an enzyme used in viral replication. The efficacyof a drug against the viral infection can be prolonged, augmented, orrestored by administering the compound in combination or alternationwith a second, and perhaps third, antiviral compound that induces adifferent mutation from that caused by the principle drug.Alternatively, the pharmacokinetics, biodistribution or other parametersof the drug can be altered by such combination or alternation therapy.In general, combination therapy is typically preferred over alternationtherapy because it induces multiple simultaneous stresses on the virus.

Any of the viral treatments described in the Background of the Inventioncan be used in combination or alternation with the compounds describedin this specification. Nonlimiting examples include:

-   -   (1) Protease inhibitors

Examples include substrate-based NS3 protease inhibitors (Attwood etal., Antiviral peptide derivatives, PCT WO 98/22496, 1998; Attwood etal., Antiviral Chemistry and Chemotherapy 1999, 10, 259-273; Attwood etal, Preparation and use of amino acid derivatives as anti-viral agents,German Patent Pub. DE 19914474; Tung et al. Inhibitors of serineproteases, particularly hepatitis C virus NS3 protease, PCT WO98/17679), including alphaketoamides and hydrazinoureas, and inhibitorsthat terminate in an electrophile such as a boronic acid or phosphonate(Llinas-Brunet et al, Hepatitis C inhibitor peptide analogues, PCT WO99/07734); Non-substrate-based NS3 protease inhibitors such as2,4,6-trihydroxy-3-nitro-benzamide derivatives (Sudo K. et al.,Biochemical and Biophysical Research Communications, 1997, 238, 643-647;Sudo K. et al. Antiviral Chemistry and Chemotherapy, 1998, 9, 186),including RD3-4082 and RD3-4078, the former substituted on the amidewith a 14 carbon chain and the latter processing a para-phenoxyphenylgroup; and Sch 68631, a phenanthrenequinone, an HCV protease inhibitor(Chu M. et al., Tetrahedron Letters 37:7229-7232, 1996).

Sch 351633, isolated from the fungus Penicillium griseofulvum, wasidentified as a protease inhibitor (Chu M. et al., Bioorganic andMedicinal Chemistry Letters 9:1949-1952). Eglin c, isolated from leech,is a potent inhibitor of several serine proteases such as S. griseusproteases A and B, α-chymotrypsin, chymase and subtilisin. Qasim M. A.et al., Biochemistry 36:1598-1607, 1997.

U.S. patents disclosing protease inhibitors for the treatment of HCVinclude, for example, U.S. Pat. No. 6,004,933 to Spruce et al. whichdiscloses a class of cysteine protease inhibitors for inhibiting HCVendopeptidase 2; U.S. Pat. No. 5,990,276 to Zhang et al. which disclosessynthetic inhibitors of hepatitis C virus NS3 protease; U.S. Pat. No.5,538,865 to Reyes et a; WO 02/008251 to Corvas International, Inc, andWO 02/08187 and WO 02/008256 to Schering Corporation. HCV inhibitortripeptides are disclosed in U.S. Pat. Nos. 6,534,523, 6,410,531, and6,420,380 to Boehringer Ingelheim and WO 02/060926 to Bristol MyersSquibb. Diaryl peptides as NS3 serine protease inhibitors of HCV aredisclosed in WO 02/48172 to Schering Corporation. Imidazolidindiones asNS3 serine protease inhibitors of HCV are disclosed in WO 02/08198 toSchering Corporation and WO 02/48157 to Bristol Myers Squibb. WO98/17679 to Vertex Pharmaceuticals and WO 02/48116 to Bristol MyersSquibb also disclose HCV protease inhibitors.

-   -   (2) Thiazolidine derivatives which show relevant inhibition in a        reverse-phase HPLC assay with an NS3/4A fusion protein and        NS5A/5B substrate (Sudo K. et al., Antiviral Research, 1996, 32,        9-18), especially compound RD-1-6250, possessing a fused        cinnamoyl moiety substituted with a long alkyl chain, RD4 6205        and RD4 6193;    -   (3) Thiazolidines and benzanilides identified in Kakiuchi N. et        al. J. EBS Letters 421, 217-220; Takeshita N. et al. Analytical        Biochemistry, 1997, 247, 242-246;    -   (4) A phenan-threnequinone possessing activity against protease        in a SDS-PAGE and autoradiography assay isolated from the        fermentation culture broth of Streptomyces sp., Sch 68631        (Chu M. et al., Tetrahedron Letters, 1996, 37, 7229-7232), and        Sch 351633, isolated from the fungus Penicillium griseofulvum,        which demonstrates activity in a scintillation proximity assay        (Chu M. et al., Bioorganic and Medicinal Chemistry Letters 9,        1949-1952);    -   (5) Helicase inhibitors (Diana G. D. et al., Compounds,        compositions and methods for treatment of hepatitis C, U.S. Pat.        No. 5,633,358; Diana G. D. et al., Piperidine derivatives,        pharmaceutical compositions thereof and their use in the        treatment of hepatitis C, PCT WO 97/36554);    -   (6) Nucleotide polymerase inhibitors and gliotoxin (Ferrari R.        et al. Journal of Virology, 1999, 73, 1649-1654), and the        natural product cerulenin (Lohmann V. et al., Virology, 1998,        249, 108-118);    -   (7) Antisense phosphorothioate oligodeoxynucleotides (S-ODN)        complementary to sequence stretches in the 5′ non-coding region        (NCR) of the virus (Alt M. et al, Hepatology, 1995, 22,        707-717), or nucleotides 326-348 comprising the 3′ end of the        NCR and nucleotides 371-388 located in the core coding region of        the HCV RNA (Alt M. et al., Archives of Virology, 1997, 142,        589-599; Galderisi U. et al., Journal of Cellular Physiology,        1999, 181, 251-257);    -   (8) Inhibitors of IRES-dependent translation (Ikeda N et al.,        Agent for the prevention and treatment of hepatitis C, Japanese        Patent Pub. JP-08268890; Kai Y. et al. Prevention and treatment        of viral diseases, Japanese Patent Pub. JP-10101591);    -   (9) Ribozymes, such as nuclease-resistant ribozymes        (Maccjak, D. J. et al., Hepatology 1999, 30, abstract 995) and        those disclosed in U.S. Pat. No. 6,043,077 to Barber et al., and        U.S. Pat. Nos. 5,869,253 and 5,610,054 to Draper et al.; and    -   (10) Nucleoside analogs have also been developed for the        treatment of Flaviviridae infections.    -   (11) Any of the compounds described by Idenix Pharmaceuticals in        International Publication Nos. WO 01/90121 and WO 01/92282;    -   (12) Other patent applications disclosing the use of certain        nucleoside analogs to treat hepatitis C virus include:        PCT/CA00/01316 (WO 01/32153; filed Nov. 3, 2000) and        PCT/CA01/00197 (WO 01/60315; filed Feb. 19, 2001) filed by        BioChem Pharma, Inc. (now Shire Biochem, Inc.); PCT/US02/01531        (WO 02/057425; filed Jan. 18, 2002) and PCT/US02/03086 (WO        02/057287; filed Jan. 18, 2002) filed by Merck & Co., Inc.,        PCT/EP01/09633 (WO 02/18404; published Aug. 21, 2001) filed by        Roche, and PCT Publication Nos. WO 01/79246 (filed Apr. 13,        2001), WO 02/32920 (filed Oct. 18, 2001) and WO 02/48165 by        Pharmasset, Ltd.    -   (13) PCT Publication No. WO 99/43691 to Emory University,        entitled “2′-Fluoronucleosides” discloses the use of certain        2′-fluoronucleosides to treat HCV.    -   (14) Other miscellaneous compounds including        1-amino-alkylcyclohexanes (U.S. Pat. No. 6,034,134 to Gold et        al.), alkyl lipids (U.S. Pat. No. 5,922,757 to Chojkier et al.),        vitamin E and other antioxidants (U.S. Pat. No. 5,922,757 to        Chojkier et al), squalene, amantadine, bile acids (U.S. Pat. No.        5,846,964 to Ozeki et al.), N-(phosphonoacetyl)-L-aspartic acid,        (U.S. Pat. No. 5,830,905 to Diana et al.), benzenedicarboxamides        (U.S. Pat. No. 5,633,388 to Diana et al.), polyadenylic acid        derivatives (U.S. Pat. No. 5,496,546 to Wang et al.),        2′,3′-dideoxyinosine (U.S. Pat. No. 5,026,687 to Yarchoan et        al.), benzimidazoles (U.S. Pat. No. 5,891,874 to Colacino et        al), plant extracts (U.S. Pat. No. 5,837,257 to Tsai et al.,        U.S. Pat. No. 5,725,859 to Omer et al., and U.S. Pat. No.        6,056,961), and piperadines (U.S. Pat. No. 5,830,905 to Diana et        al.).    -   (15) Any other compound currently in preclinical or clinical        development for treatment of hepatitis C virus including:        Interleukin-10 by Schering-Plough, IP-501 by Interneuron,        Merimebodib (VX-497) by Vertex, AMANTADINE® (Symmetrel) by Endo        Labs Solvay, HEPTAZYME® by RPI, IDN-6556 by Idun Pharma.,        XTL-002 by XTL., HCV/MF59 by Chiron, CIVACIR (Hepatitis C Immune        Globulin) by NABI, LEVOVIRIN® by ICN/Ribapharm, VIRAMIDINE® by        ICN/Ribaphamm, ZADAXIN® (thymosin alpha-1) by Sci Clone,        thymosin plus pegylated interferon by Sci Clone, CEPLENE®        (histamine dihydrochloride) by Maxim, VX 950/LY 570310 by        Vertex/Eli Lilly, ISIS 14803 by Isis Pharmaceutical/Elan,        IDN-6556 by Idun Pharmaceuticals, Inc., JTK 003 by AKROS Pharma,        BILN-2061 by Boehringer Ingelheim, CellCept (mycophenolate        mofetil) by Roche, T67, a β-tubulin inhibitor, by Tularik, a        therapeutic vaccine directed to E2 by Innogenetics, FK788 by        Fujisawa Healthcare, Inc., 1 dB 1016 (Siliphos, oral        silybin-phosphatdylcholine phytosome), RNA replication        inhibitors (VP50406) by ViroPharma/Wyeth, therapeutic vaccine by        Intercell, therapeutic vaccine by Epimmune/Genencor, IRES        inhibitor by Anadys, ANA 245 and ANA 246 by Anadys,        immunotherapy (Therapore) by Avant, protease inhibitor by        Corvas/SChering, helicase inhibitor by Vertex, fusion inhibitor        by Trimeris, T cell therapy by CellExSys, polymerase inhibitor        by Biocryst, targeted RNA chemistry by PTC Therapeutics,        Dication by Immtech, Int., protease inhibitor by Agouron,        protease inhibitor by Chiron/Medivir, antisense therapy by AVI        BioPharma, antisense therapy by Hybridon, hemopurifier by        Aethlon Medical, therapeutic vaccine by Merix, protease        inhibitor by Bristol-Myers Squibb/Axys, Chron-VacC, a        therapeutic vaccine, by Tripep, UT 231B by United Therapeutics,        protease, helicase and polymerase inhibitors by Genelabs        Technologies, IRES inhibitors by Immusol, R803 by Rigel        Pharmaceuticals, INFERGEN® (interferon alphacon-1) by InterMune,        OMNIFERON® (natural interferon) by Viragen, ALBUFERON® by Human        Genome Sciences, REBIF (interferon beta-1a) by Ares-Serono,        Omega Interferon by BioMedicine, Oral Interferon Alpha by        Amarillo Biosciences, interferon gamma, interferon tau, and        Interferon gamma-1b by InterMune.        V. Biological Data        Cell Culture Systems for Determining Antiviral Activities

A useful cell-based assay to detect HCV and its inhibition assesses thelevels of replicon RNA from Huh7 cells harboring the HCV replicon. Thesecells can be cultivated in standard media, for example DMEM medium (highglucose, no pyruvate), supplemented with 10% fetal bovine serum, 1×non-essential amino acids, Pen-Strep-Glu (100 units/liter, 100microgram/liter, and 2.92 mg/liter, respectively), and G418(C₂₀H₄₀N₄O₁₀; 500 to 1000 microgram/milliliter). Antiviral screeningassays can be done in the same medium without G418. To keep the cells inthe logarithmic growth phase, cells are seeded in 96-well plates at lowdensity (for example, 1000 cells per well). The test compound is thenadded immediately after seeding the cells and they are incubated for 3to 7 days at 37° C. in an incubator. The medium is then removed, and thecells prepared for total RNA extraction (replicon RNA+host RNA).Replicon RNA can then be amplified in a real-time RT-PCR (Q-RT-PCR)protocol, and quantified.

The observed differences in quantification of replicon RNA are one wayto express the antiviral potency of the test compound. In a typicalexperiment, a comparable amount of replicon is produced in the negativecontrol and with non-active compounds. This can be concluded if themeasured threshold-cycle for HCV RT-PCR in both setting is approximatelythe same. In such experiments, a way to express the antiviraleffectiveness of a compound is to subtract the average threshold RT-PCRcycle of the negative control (Ct_(negative)) from the threshold RT-PCRcycle of the test compound (Ct_(testcompound)). This value is called ΔCt(ΔCt=Ct_(testcompound)−Ct_(negative)). A ΔCt value of 3.3 represents a1-log reduction in replicon production. As a positive control,recombinant interferon alpha-2a (for example, Roferon-A, Hoffmann-Roche,N.J., USA) can be used alongside the test compound. Furthermore, thecompounds can be tested in dilution series (typically at 100, 33, 10, 3and 1 μM). The ΔCt values for each concentration allow the calculationof the 50% effective concentration (EC₅₀).

The assay described above can be adapted to the other members of theFlaviviridae by changing the cell system and the viral pathogen.Methodologies to determine the efficacy of these antiviral compoundsinclude modifications of the standard techniques as described byHolbrook M R et al. Virus Res. 2000, 69, 31; Markland W et al.Antimicrob. Agents. Chemother. 2000, 44, 859; Diamond M S et al., J.Virol. 2000, 74, 7814; Jordan I et al. J. Infect. Dis. 2000, 182, 1214;Sreenivasan V et al. J. Virol. Methods 1993, 45 (1), 1; or Baginski S Get al. Proc. Natl. Acad. Sci. U.S.A. 2000, 97 (14), 7981 or thereal-time RT-PCR technology. As an example, an HCV replicon system inHuH7 cells (Lohmann V et al. Science. 1999, 285 (5424), 110) ormodifications thereof (Blight et al. 2000) can be used.

Non-Cell Based Assays Adapted for Detecting HCV

Nucleic acid amplification technology is now the method of choice foridentification of a large and still growing number of microorganismssuch as Mycobacterium tuberculosis, human immunodeficiency virus (HIV),and hepatitis C virus (HCV) in biological samples. Nucleic acidamplification techniques include the polymerase chain reaction (PCR),ligase chain reaction (LCR), nucleic acid sequence-based amplification(NASBA), strand-displacement amplification (SDA), andtranscription-mediated amplification (TMA). Several FDA-approveddiagnostic products incorporate these molecular diagnostic methods (seeTable below). Nucleic acid amplification technology tests involve notonly amplification, but detection methodologies as well. The promise ofmolecular diagnostics lies in the improvement of itsspecimen-processing, amplification, and target-detection steps, and inthe integration of these steps into an automated format.

Amplification Product: Nucleic Acid FDA- Approved Assays DetectionMethod Amplification Method Commercial Source C. trachomatis,Heterogeneous: PCR Roche Diagnostics N. gonorrhoeae, Colorimetric M.tuberculosis, HIV-1 C. trachomatis, Heterogeneous: LCR AbbottLaboratories N. gonorrhoeae Chemiluminescence C. trachomatis,Homogeneous: SDA Becton Dickinson N. gonorrhoeae Fluorescence C.trachomatis, Homogeneous: TMA Gen-Probe M. tuberculosisChemiluminescence (HPA)Amplified-Product Detection Schemes

Amplified-product detection schemes are of two basic types:heterogeneous and homogeneous. Heterogeneous detection is characterizedby a distinct step, such as washing, designed to remove unhybridizedprobes from hybridized probes, whereas in homogeneous detection there isno physical separation step to remove free probe from bound probe.Multiple heterogeneous and homogeneous detection methods exist. Any ofthese heterogeneous or homogeneous assays can be utilized to assess theeffectiveness of the compounds of the present invention versus anRNA-dependent RNA polymerase virus, such as HCV.

Heterogeneous Detection: Southern blotting, for example, is aheterogeneous detection technique. In Southern blotting, electrophoresisis used to separate amplification products by size and charge. Thesize-fractionated products are transferred to a membrane or filter bydiffusion, vacuuming, or electroblotting. Labeled detection probes arethen hybridized to the membrane-bound targets in solution, the filtersare washed to remove any unhybridized probe, and the hybridized probe onthe membrane is detected by any of a variety of methods.

Other types of heterogeneous detection are based on specific capture ofthe amplification products by means of enzyme-linked immunosorbentassays (ELISAs). One method used with PCR involves labeling one primerwith a hapten or a ligand, such as biotin, and, after amplification,capturing it with an antibody- or streptavidin-coated microplate. Theother primer is labeled with a reporter such as fluorescein, anddetection is achieved by adding an antifluorescein antibody, horseradishperoxidase (HRP) conjugate. This type of method is not as specific asusing detection probes that hybridize to defined amplification productsof interest.

The LCx probe system (Abbott Laboratories; Abbott Park, Ill.) and theAmplicor HIV-1 test (Roche Molecular Systems Inc.; Pleasanton, Calif.)are systems that use heterogeneous detection methods. In the LCx system,hapten-labeled oligonucleotide probes thermocycle in the ligase chainreaction. Either a capture hapten or a detection hapten is covalentlyattached to each of the four primer oligonucleotides. Uponamplification, each amplified product (amplicon) has one capture haptenand one detection hapten. When amplification is complete, the LCx systeminstrument transfers the reaction to a new well where antibody-coatedmicroparticles bind the capture haptens. Each microparticle is thenirreversibly bound to a glass-fiber matrix. A wash step removes from themicroparticle any probe that contains only the detection hapten. The LCxinstrument adds an alkaline phosphatase (AP)-antibody conjugate thatbinds to the detection hapten. A fluorigenic substrate for AP is4-methylumbelliferyl. Dephosphorylation of 4-methylumbelliferyl by APconverts it to 4-methylumbelliferone, which is fluorescent.

The Amplicor HIV-1 test uses an ELISA format. After amplification byPCR, the amplicon is chemically denatured. Amplicon-specificoligonucleotide probes capture the denatured strands onto a coatedmicroplate. The operator washes away any unincorporated primers andunhybridized material in a wash step and then adds an avidin-HRPconjugate to each well. The conjugate binds to the biotin-labeledamplicon captured on the plate. The operator then adds3,3′,5,5′-tetramethylbenzidine (TMB), a chromogenic HRP substrate. Whenhydrogen peroxide is present, HRP oxidizes TMB. The signal is determinedcolorimetrically.

Homogeneous Detection: Because hybridized and nonhybridized detectionprobes are not physically separated in homogeneous detection systems,these methods require fewer steps than heterogeneous methods and thusare less prone to contamination. Among the commercially available kitsthat use homogeneous detection of fluorescent and chemiluminescentlabels are the TaqMan system (Applied Biosystems; Foster City, Calif.),BDProbeTecET system (Becton Dickinson; Franklin Lakes, N.J.), QPCRSystem 5000 (Perkin-Elmer Corp.; Norwalk, Conn.), and HybridizationProtection Assay (Gen-Probe Inc.; San Diego).

The TaqMan system detects amplicon in real time. The detection probe,which hybridizes to a region inside the amplicon, contains a donorfluorophore such as fluoroscein at its 5′ end and a quencher moiety, forexample, rhodamine, at its 3′ end. When both quencher and fluorophoreare on the same oligonucleotide, donor fluorescence is inhibited. Duringamplification the probe is bound to the target. Taq polymerase displacesand cleaves the detection probe as it synthesizes the replacementstrand. Cleavage of the detection probe results in separation of thefluorophore from the quencher, leading to an increase in the donorfluorescence signal. During each cycle of amplification the process isrepeated. The amount of fluorescent signal increases as the amount ofamplicon increases.

Molecular beacons use quenchers and fluorophores also. Beacons areprobes that are complementary to the target amplicon, but contain shortstretches (approximately 5 nucleotides) of complementaryoligonucleotides at each end. The 5′ and 3′ ends of the beacons arelabeled with a fluorophore and a quencher, respectively. A hairpinstructure is formed when the beacon is not hybridized to a target,bringing into contact the fluorophore and the quencher and resulting influorescent quenching. The loop region contains the region complementaryto the amplicon. Upon hybridization to a target, the hairpin structureopens and the quencher and fluorophore separate, allowing development ofa fluorescent signal.¹⁴ A fluorometer measures the signal in real time.

The BDProbeTecET system uses a real-time detection method that combinesaspects of TaqMan and molecular beacons. The probe has a hairpin loopstructure and contains fluorescein and rhodamine labels. In this system,however, the region complementary to the target molecule is not withinthe loop but rather in the region 3′ to the rhodamine label. Instead ofcontaining the sequence complementary to the target, the single-strandedloop contains a restriction site for the restriction enzyme BsoBI. Thesingle-stranded sequence is not a substrate for the enzyme. Thefluorescein and rhodamine labels are near each other beforeamplification, which quenches the fluorescein fluorescence.Strand-displacement amplification converts the probe into adouble-stranded molecule. The BsoBI restriction enzyme can then cleavethe molecule, resulting in separation of the labels and an increase inthe fluorescent signal.

The QPCR System 5000 employs electrochemiluminescence with rutheniumlabels. A biotinylated primer is used. After amplification, the biotinproducts are captured on streptavidin-coated paramagnetic beads. Thebeads are transferred into an electrochemical flow cell by aspirationand magnetically held to the surface of the electrode. Upon electricalstimulation, the ruthenium-labeled probe emits light.

The Hybridization Protection Assay is used in Gen-Probe's nonamplifiedPACE assays as well as in amplified Mycobacterium tuberculosis andChlamydia trachomatis assays. The detection oligonucleotide probes inHPA are labeled with chemiluminescent acridinium ester (AE) by means ofa linker arm. Hybridization takes place for 15 minutes at 60° C. in thesame tube in which the amplification occurred. The selection reagent, amildly basic buffered solution added after hybridization, hydrolyzes theAE on any unhybridized probe, rendering it nonchemiluminescent. The AEon hybridized probes folds inside the minor groove of the double helix,thereby protecting itself from hydrolysis by the selection reagent. TheAE emits a chemiluminescent signal upon hydrolysis by hydrogen peroxidefollowed by sodium hydroxide. A luminometer records the chemiluminescentsignal for 2 seconds (a period termed a light-off) and reports thephotons emitted in terms of relative light units (RLU).

Detection-probe design is critical in all methodologies that use probesto detect amplification products. Good detection probes hybridize onlyto specified amplification product and do not hybridize to nonspecificproducts. Other key issues in optimizing detection methodologies involvethe labeling of probes and the maximization of sample throughput.

Labeling Methods and Reporter Molecules. Detection probes can be labeledseveral different ways. Enzymatic incorporation of ³²P or ³⁵S into theprobes is the most common method for isotopic labeling. Followinghybridization and washing, the signal is detected on autoradiographicfilm.

To perform nonradioactive detection, probes can be enzymatically labeledwith a variety of molecules. Biotin can be incorporated enzymaticallyand then detected with streptavidin-conjugated alkaline phosphatase,using AP substrates like 5-bromo-4-chloro-3-indolyl phosphate (BCIP) andnitroblue tetrazolium (NBT). Chemiluminescent substrates such asLumi-Phos 530 or Lumi-Phos Plus (Lumigen, Southfield, Mich.) can also beused with AP. In addition, digoxigenin-11-dUTP can be incorporatedenzymatically into DNA or RNA, and antidigoxigenin AP conjugates can beused with calorimetric or chemiluminescent detection.

There are numerous other types of reporter molecules, includingchemiluminescent moieties such as acridinium esters. Many fluorescentmoieties are available as well. Electrochemiluminescent compounds suchas tris(2,2′-bipyridine) ruthenium (II) can be used also. Furtherdiscussions of these and similar techniques can be found in: Schiff E R,de Medina M, Kahn R S. Semin Liver Dis. 1999; 19(Suppl 1:3-15).

EXAMPLE 3 Cellular Pharmacology of 2′-C-methyl-cytidine-3′-O-L-valineester (val-mCyd)

Phosphorylation Assay of Nucleoside to Active Triphosphate

To determine the cellular metabolism of the compounds, HepG2 cells areobtained from the American Type Culture Collection (Rockville, Md.), andare grown in 225 cm² tissue culture flasks in minimal essential mediumsupplemented with non-essential amino acids, 1% penicillin-streptomycin.The medium is renewed every three days, and the cells are subculturedonce a week. After detachment of the adherent monolayer with a 10 minuteexposure to 30 mL of trypsin-EDTA and three consecutive washes withmedium, confluent HepG2 cells are seeded at a density of 2.5×10⁶ cellsper well in a 6-well plate and exposed to 10 μM of [³H] labeled activecompound (500 dpm/pmol) for the specified time periods. The cells aremaintained at 37° C. under a 5% CO₂ atmosphere. At the selected timepoints, the cells are washed three times with ice-coldphosphate-buffered saline (PBS). Intracellular active compound and itsrespective metabolites are extracted by incubating the cell pelletovernight at −20° C. with 60% methanol followed by extraction with anadditional 20 μL of cold methanol for one hour in an ice bath. Theextracts are then combined, dried under gentle filtered air flow andstored at −20° C. until HPLC analysis.

Antiviral nucleosides and nucleoside analogs are generally convertedinto the active metabolite, the 5′-triphosphate (TP) derivatives byintracellular kinases. The nucleoside-TPs then exert their antiviraleffect by inhibiting the viral polymerase during virus replication. Inprimary human hepatocyte cultures, in a human hepatoma cell line(HepG2), and in a bovine kidney cell line (MDBK), mCyd was convertedinto a major metabolite, 2′-C-methyl-cytidine-5′-triphosphate (mCyd-TP),along with smaller amounts of a uridine 5′-triphosphate derivative,2′-C-methyl-uridine-5′-triphosphate (mUrd-TP). mCyd-TP is inhibitorywhen tested in vitro against the BVDV replication enzyme, the NS5B RNAdependent RNA polymerase, and was thought to be responsible for theantiviral activity of mCyd.

The cellular metabolism of mCyd was examined using MDBK cells, HepG2cells and human primary hepatocytes exposed to 10 μM [³H]-mCyd.High-pressure liquid chromatography (HPLC) analysis demonstrated thatmCyd was phosphorylated in all three cell types, with mCyd-TP being thepredominant metabolite after 24 h. The metabolic profile obtained over a48-hour exposure of human hepatoma HepG2 cells to 10 μM [³H]-mCyd wastested. In HepG2 cells, levels of mCyd-TP peaked at 41.5±13.4 μM after24 hours (see Table 3) and fell slowly thereafter. In primary humanhepatocytes, the peak mCyd-TP concentration at 24 hours was 4 fold lowerat 10.7±6.7 μM. MDBK bovine kidney cells yielded intermediate levels ofmCyd-TP (30.1±6.9 μM at 24 hours).

Exposure of hepatocytes to mCyd led to production of a second5′-triphosphate derivative, mUrd-TP. In HepG2 cells exposed to 10 μM[³H]-mCyd, the mUrd-TP level reached 1.9±1.6 μM at 24 hours, compared to8.1±3.4 μM in MDBK cells and 3.2±2.0 μM in primary human hepatocytes.While MDBK and HepG2 cells produced comparable total amounts ofphosphorylated species (approximately 43 versus 47 μM, respectively) at24 h, mUrd-TP comprised 19% of the total product in MDBK cells versusonly 4% in HepG2 cells. mUrd-TP concentration increased steadily overtime, however reached a plateau or declined after 24 hours.

TABLE 3 Activation of mCyd (10 μM) in Hepatocytes and MDBK CellsMetabolite (μM) Cells^(a) n mCyd-MP mUrd-MP mCyd-DP mUrd-DP mCyd-TPmUrd-TP HepG2 6 ND ND 3.7 ± 2.1 ND  41.5 ± 13.4 1.9 ± 1.6 Human 5 ND ND1.15 ± 1.1   0.26 ± 0.4 C 10.7 ± 6.7 3.2 ± 2.0 Primary Hepatocytes MDBKBovine 7 ND ND 4.2 ± 2.7 0.76 ± 0.95 30.1 ± 6.9 8.1 ± 3.4 Kidney Cells^(a)Cells were incubated for 24 hours with [³H]-mCyd, specific activity:HepG2 assay = 0.5 Ci/mmol; human and monkey hepatocyte assay = 1.0Ci/mmol. ^(b)The concentrations of metabolites were determined as pmolesper million cells. One pmole per million cells is roughly equivalent to1 μM. ND, not detected.

The apparent intracellular half-life of the mCyd-TP was 13.9±2.2 hoursin HepG2 cells and 7.6±0.6 hours in MDBK cells: the data were notsuitable for calculating the half life of mUrd-TP. Other than thespecific differences noted above, the phosphorylation pattern detectedin primary human hepatocytes was qualitatively similar to that obtainedusing HepG2 or MDBK cells.

EXAMPLE 4 Cell Cytotoxicity

Mitochondria Toxicity Assay

HepG2 cells were cultured in 12-well plates as described above andexposed to various concentrations of drugs as taught by Pan-Zhou X-R,Cui L, Zhou X-J, Sommadossi J-P, Darley-Usmer V M. “Differential effectsof antiretroviral nucleoside analogs on mitochondrial function in HepG2cells” Antimicrob. Agents Chemother. 2000; 44:496-503. Lactic acidlevels in the culture medium after 4 day drug exposure were measuredusing a Boehringer lactic acid assay kit. Lactic acid levels werenormalized by cell number as measured by hemocytometer count.

Cytotoxicity Assays

Cells were seeded at a rate of between 5×10³ and 5×10⁴/well into 96-wellplates in growth medium overnight at 37° C. in a humidified CO₂ (5%)atmosphere. New growth medium containing serial dilutions of the drugswas then added. After incubation for 4 days, cultures were fixed in 50%TCA and stained with sulforhodamineB. The optical density was read at550 nm. The cytotoxic concentration was expressed as the concentrationrequired to reduce the cell number by 50% (CC₅₀).

Conventional cell proliferation assays were used to assess thecytotoxicity of mCyd and its cellular metabolites in rapidly dividingcells. The inhibitory effect of mCyd was determined to be cytostatic innature since mCyd showed no toxicity in confluent cells atconcentrations far in excess of the corresponding CC₅₀ for a specificcell line. mCyd was not cytotoxic to rapidly growing Huh7 human hepatomacells or H9c2 rat myocardial cells at the highest concentration tested(CC₅₀>250 μM). The mCyd CC₅₀ values were 132 and 161 μM in BHK-21hamster kidney and HepG2 human hepatoma cell lines, respectively. TheCC₅₀ for mCyd in HepG2 cells increased to 200 μM when the cells weregrown on collagen-coated plates for 4 or 10 days. For comparison, CC₅₀values of 35-36 μM were derived when ribavirin was tested in HepG2 andHuh7 cells. In the MDBK bovine kidney cells used for BVDV antiviralstudies, the CC₅₀ of mCyd was 36 μM. A similar CC₅₀ value (34 μM) wasdetermined for mCyd against MT-4 human T-lymphocyte cells. In addition,mCyd was mostly either non-cytotoxic or weakly cytotoxic (CC₅₀>50to >200 μM) to numerous other cell lines of human and other mammalianorigin, including several human carcinoma cell lines, in testingconducted by the National Institutes of Health (NIH) Antiviral Researchand Antimicrobial Chemistry Program. Exceptions to this were rapidlyproliferating HFF human foreskin fibroblasts and MEF mouse embryofibroblasts, where mCyd showed greater cytotoxicity (CC₅₀s 16.9 and 2.4μM, respectively). Again, mCyd was much less toxic to stationary phasefibroblasts.

The cytotoxic effect of increasing amounts of mCyd on cellular DNA orRNA synthesis was examined in HepG2 cells exposed to [³H]-thymidine or[³H]-uridine. In HepG2 cells, the CC₅₀s of mCyd required to cause 50%reductions in the incorporation of radiolabeled thymidine and uridineinto cellular DNA and RNA, were 112 and 186 μM, respectively. The CC₅₀values determined for ribavirin (RBV) for DNA and RNA synthesis,respectively, were 3.16 and 6.85 μM. These values generally reflect theCC₅₀s of 161 and 36 μM determined for mCyd and RBV, respectively, inconventional cell proliferation cytotoxicity assays. To assess theincorporation of mCyd into cellular RNA and DNA, HepG2 cells wereexposed to 10 μM [³H]-mCyd or control nucleosides (specific activity5.6-8.0 Ci/mmole, labeled in the base) for 30 hours. Labeled cellularRNA or DNA species were separately isolated and incorporation wasdetermined by scintillation counting. Exposure of HepG2 cells to mCydresulted in very low levels of incorporation of the ribonucleosideanalog into either cellular DNA or RNA (0.0013-0.0014 pmole/μg ofnucleic acid). These levels are similar to the 0.0009 and 0.0013 pmole/1g values determined for the incorporation of ZDV and ddC, respectively,into RNA: since these deoxynucleosides are not expected to incorporateinto RNA, these levels likely reflect the assay background. Theincorporation of ZDV and ddC into DNA was significantly higher (0.103and 0.0055 pmole/μg, respectively). Ribavirin (RBV) incorporated intoboth DNA and RNA at levels 10-fold higher than mCyd.

TABLE 4a Cellular Nucleic Acid Synthesis and Incorporation Studies inHepG2 Cells (10 μM Drug and Nucleoside Controls) CC₅₀ (μM) DNA RNAIncorporated drug amount Compound ([³H]Thymidine) ([³H]Uridine) pmole/μgDNA pmole/μg RNA mCyd 112.3 ± 34.5 186.1 ± 28.2 0.0013 ± 0.0008^(a)0.0014 ± 0.0008^(a) ZDV nd nd  0.103 ± 0.0123^(a) 0.0009 ± 0.0003^(a)ddC nd nd 0.0055^(b) 0.0013^(b) Ribavirin  3.16 ± 0.13  6.85 ± 1.830.0120^(b) 0.0132^(c) ^(a)Data represent mean of three experiments^(b)Data represent one experiment ^(c)Data represent mean of twoexperiments nd, not determined

TABLE 4b Cytotoxicity of mCyd in Mammalian Cell Lines Cell Line^(a) nCC₅₀ (μM) Huh 7 7 >250 Hep G2 6 161 ± 19 Hep G2^(b) 2 >200 MDBK 7 36 ± 7BHK-21 2 132 ± 6  H9c2 2 >250 ^(a)All cytotoxicity testing was doneunder conditions of rapid cell division ^(b)Cells were grown on collagencoated plates for 4 or 10 dEffect on Human Bone Marrow Progenitor CellsBone Marrow Toxicity Assay

Human bone marrow cells were collected from normal healthy volunteersand the mononuclear population was separated by Ficoll-Hypaque gradientcentrifugation as described previously by Sommadossi J-P, Carlisle R.“Toxicity of 3′-azido-3′-deoxythymidine and9-(1,3-dihydroxy-2-propoxymethyl)guanine for normal human hematopoieticprogenitor cells in vitro” Antimicrobial Agents and Chemotherapy 1987;31:452-454; and Sommadossi J-P, Schinazi R F, Chu C K, Xie M-Y.“Comparison of cytotoxicity of the (−)- and (+)-enantiomer of2′,3′-dideoxy-3′-thiacytidine in normal human bone marrow progenitorcells” Biochemical Pharmacology 1992; 44:1921-1925. The culture assaysfor CFU-GM and BFU-E were performed using a bilayer soft agar ormethylcellulose method. Drugs were diluted in tissue culture medium andfiltered. After 14 to 18 days at 37° C. in a humidified atmosphere of 5%CO₂ in air, colonies of greater than 50 cells were counted using aninverted microscope. The results are presented as the percent inhibitionof colony formation in the presence of drug compared to solvent controlcultures.

Cell Protection Assay (CPA)

The assay was performed essentially as described by Baginski, S. G.;Pevear, D. C.; Seipel, M.; Sun, S. C. C.; Benetatos, C. A.; Chunduru, S.K.; Rice, C. M. and M. S. Collett “Mechanism of action of a pestivirusantiviral compound” PNAS USA 2000, 97(14), 7981-7986. MDBK cells (ATCC)were seeded onto 96-well culture plates (4,000 cells per well) 24 hoursbefore use. After infection with BVDV (strain NADL, ATCC) at amultiplicity of infection (MOI) of 0.02 plaque forming units (PFU) percell, serial dilutions of test compounds were added to both infected anduninfected cells in a final concentration of 0.5% DMSO in growth medium.Each dilution was tested in quadruplicate. Cell densities and virusinocula were adjusted to ensure continuous cell growth throughout theexperiment and to achieve more than 90% virus-induced cell destructionin the untreated controls after four days post-infection. After fourdays, plates were fixed with 50% TCA and stained with sulforhodamine B.The optical density of the wells was read in a microplate reader at 550nm. The 50% effective concentration (EC₅₀) values are defined as thecompound concentration that achieved 50% reduction of cytopathic effectof the virus.

The myelosuppressive effects of certain nucleoside analogs havehighlighted the need to test for potential effects of investigationaldrugs on the growth of human bone marrow progenitor cells in clonogenicassays. In particular, anemia and neutropenia are the most commondrug-related clinical toxicities associated with the anti-HIV drugzidovudine (ZDV) or the ribavirin (RBV) component of the standard ofcare combination therapy used for HCV treatment. These toxicities havebeen modeled in an in vitro assay that employed bone marrow cellsobtained from healthy volunteers (Sommadossi J-P, Carlisle R.Antimicrob. Agents Chemother. 1987; 31(3): 452-454). ZDV has beenpreviously shown to directly inhibit human granulocyte-macrophagecolony-forming (CFU-GM) and erythroid burst-forming (BFU-E) activity atclinically relevant concentrations of 1-2 μM in this model (Berman E, etal. Blood 1989; 74(4):1281-1286; Yoshida Y, Yoshida C. AIDS Res. Hum.Retroviruses 1990; 6(7):929-932.; Lerza R, et al. Exp. Hematol. 1997;25(3):252-255; Domsife R E, Averett D R. Antimicrob. Agents Chemother.1996; 40(2):514-519; Kurtzberg J, Carter S G. Exp. Hematol. 1990;18(10):1094-1096; Weinberg R S, et al. Mt. Sinai. J. Med 1998;65(1):5-13). Using human bone marrow clonogenic assays, the CC₅₀ valuesof mCyd in CFU-GM and BFU-E were 14.1±4.5 and 13.9±3.2 μM (see Table 5).mCyd was significantly less toxic to bone marrow cells than both ZDV andRBV (Table 5).

TABLE 5 Bone Marrow Toxicity of mCyd in Granulocyte MacrophageProgenitor and Erythrocyte Precursor Cells CFU-GM^(a) BFU-E^(a) CompoundCC₅₀ (μM) CC₅₀ (μM) mCyd 14.1 ± 4.5 μM 13.9 ± 3.2 ZDV 0.89 ± 0.47 0.35 ±0.28 RBV 7.49 ± 2.20 0.99 ± 0.24 ^(a)Data from 3 independent experimentsfor RBV and 5-8 independent experiments for mCyd and ZDV. Allexperiments were done in triplicate.Effect on Mitochondrial Function

Antiviral nucleoside analogs approved for HIV therapy such as ZDV,stavudine (d4T), didanosine (ddI), and zalcitabine (ddC) have beenoccasionally associated with clinically limiting delayed toxicities suchas peripheral neuropathy, myopathy, and pancreatitis (Browne M J, et al.J. Infect. Dis. 1993; 167(1):21-29; Fischl M A, et al. Ann. Intern. Med.1993; 18(10):762-769.; Richman D D, et al. N. Engl. J. Med 1987; 317(4):192-197; Yarchoan R, et al. Lancet 1990; 336(8714):526-529). Theseclinical adverse events have been attributed by some experts toinhibition of mitochondrial function due to reduction in mitochondrialDNA (mtDNA) content and nucleoside analog incorporation into mtDNA. Inaddition, one particular nucleoside analog, fialuridine(1,-2′-deoxy-2′-fluoro-1-β-D-arabinofuranosyl-5-iodo-uracil;FIAU)—caused hepatic failure, pancreatitis, neuropathy, myopathy andlactic acidosis due to direct mitochondrial toxicity (McKenzie R, et al.N Engl J Med 1995; 333(17):1099-1105). Drug-associated increases inlactic acid production can be considered a marker of impairedmitochondrial function or oxidative phosphorylation. (Colacino, J. M.Antiviral Res 1996 29(2-3): 125-39).

To assess the potential of mCyd to produce mitochondrial toxicity,several in vitro studies were conducted using the human hepatoma celllines HepG2 or Huh7. These studies included analysis of lactic acidproduction, mtDNA content, and determination of changes in morphology(e.g., loss of cristae, matrix dissolution and swelling, and lipiddroplet formation) of mitochondrial ultrastructure.

The effects of mCyd on mitochondria are presented in Table 6. Nodifferences were observed in lactic acid production in mCyd-treatedcells versus untreated cells at up to 50 μM mCyd in Huh7 cells or 10 μMmCyd in HepG2 cells. A modest (38%) increase in lactic acid productionwas seen in HepG2 cells treated with 50 μM mCyd. The significance ofthis finding is unclear, particularly since mCyd is unlikely to attain aplasma concentration of 50 μM in the clinic. For comparison, lactic acidproduction increases by 100% over control cells in cells treated with 10μM FIAU (Cui L, Yoon, et al. J. Clin. Invest. 1995; 95:555-563).Exposure of HepG2 cells to mCyd for 6 or 14 days at concentrations up to50 μM had no negative effect on mitochondrial DNA content compared to a56 or 80% reduction in ddC-treated cells, respectively.

Following 14 days of exposure to 10 μM mCyd, the ultrastructure of HepG2cells, and in particular mitochondria, was examined by transmissionelectron microscopy. No changes in cell architecture, or inmitochondrial number or morphology (including cristae), were observed inthe majority of cells. In 17% of the cells, 1 to 2 mitochondria out ofan average of 25 per cell appeared enlarged. Such minor changes would beunlikely to have any significant impact on mitochondrial function.ddC-treated cells showed abnormal mitochondrial morphology with loss ofcristae, and the accumulation of fat droplets. (Medina, D. J., C. H.Tsai, et al. Antimicrob. Agents Chemother. 1994 38(8): 1824-8; Lewis W,et al. J. Clin. Invest. 1992; 89(4):1354-1360., Lewis, L. D., F. M.Hamzeh, et al. Antimicrob. Agents Chemother. 1992 36(9): 2061-5).

TABLE 6 Effect of mCyd on Hepatocyte Proliferation. MitochondrialFunction, and Morphology in HepG2 Cells L-Lactate mtDNA/nuclear DNAElectron (% of Control^(a)) (% of Control^(b)) Microscopy^(c) Conc HepG2Huh7 6 day 14 day Lipid Mito. Agent (μM) Cells Cells Treatment TreatmentDroplet Form. Morphol. Cont. 0 100 100 100 100 Negative Normal mCyd 10 98.6 ± 7.3 98.0 ± 12.3 117.3 ± 17.5 99.7 ± 23.9 Negative Normal^(d) 50138.0 ± 8.9 97.1 ± 10.1 158.2 ± 17.5 83.0 ± 15.5 nd nd ddC 1 nd nd 44.3± 9.3 19.6 ± 8.2  nd nd 10 nd nd nd nd Positive Loss of CristaeEffect on Human DNA Polymerases α, β, and γ

The cellular DNA polymerases are responsible for normal nuclear andmitochondrial DNA synthesis and repair. Nucleoside analog triphosphatesare potential inhibitors of DNA polymerases and hence could disruptcritical cell functions. In particular, the inhibition of humanpolymerase γ, the enzyme responsible for mitochondrial DNA synthesis,has been linked to defects in mitochondrial function (Lewis, W., E. S.Levine, et al. Proceedings of the National Academy of Sciences, USA 199693(8): 3592-7). Experiments were undertaken to determine if mCyd-TPinhibited human DNA polymerases. As shown in Table 7 mCyd-TP was not asubstrate for human DNA polymerases α, β, or γ. Even 1 mM mCyd-TP failedto inhibit these enzymes by 50% in the majority of replicate assays andIC₅₀ values could only be determined to be in excess of 880-1000 μM. Incontrast, ddC was a potent inhibitor of all three human DNA polymerasesand of polymerases β and γ in particular (IC₅₀s of 4.8 and 2.7 μM,respectively). Potent inhibition was also seen for the control drug,actinomycin D, a known inhibitor of DNA-dependent-DNA polymerases.

TABLE 7 Inhibition of Human Polymerases by mCyd-Triphosphate IC₅₀ (μM)mCyd-TP^(a) ddC-TP^(b) Act. D^(a) Pol α >1000   78 ± 23.4   5.8 ± 3.1Pol β >883.3 ± 165 4.8 ± 1 7.9 ± 3 Pol γ >929.3 ± 100 2.7 ± 1 15.5 ± 4 ^(a)Mean ± S.D. from 4 data sets ^(b)Mean ± S.D. from 2 data sets

-   -   a. HepG2 or huh7 cells were treated with compounds for 4 days,        data represent at least three independent experiments    -   b. HepG2 cells were treated with compounds for 6 and 14 days,        data represents at least three independent experiments    -   c. HepG2 cells were treated with compounds for 14 days    -   d. 17% cell (11 of 64) contained 1 or 2 enlarged mitochondria        out of 25 in two independent experiments nd, not determined

EXAMPLE 5 In Vitro Antiviral Activity Against BVDV

Compounds can exhibit anti-flavivirus or pestivirus activity byinhibiting flavivirus or pestivirus polymerase, by inhibiting otherenzymes needed in the replication cycle, or by other pathways.

Plaque Reduction Assay

For each compound the effective concentration was determined induplicate 24-well plates by plaque reduction assays. Cell monolayerswere infected with 100 PFU/well of virus. Then, serial dilutions of testcompounds in MEM supplemented with 2% inactivated serum and 0.75% ofmethyl cellulose were added to the monolayers. Cultures were furtherincubated at 37° C. for 3 days, then fixed with 50% ethanol and 0.8%Crystal Violet, washed and air-dried. Then plaques were counted todetermine the concentration to obtain 90% virus suppression.

Yield Reduction Assay

For each compound the concentration to obtain a 6-log reduction in viralload was determined in duplicate 24-well plates by yield reductionassays. The assay was performed as described by Baginski, S. G.; Pevear,D. C.; Seipel, M.; Sun, S. C. C.; Benetatos, C. A.; Chunduru, S. K.;Rice, C. M. and M. S. Collett “Mechanism of action of a pestivirusantiviral compound” PNAS USA 2000, 97(14), 7981-7986, with minormodifications. Briefly, MDBK cells were seeded onto 24-well plates(2×105 cells per well) 24 hours before infection with BVDV (NADL strain)at a multiplicity of infection (MOI) of 0.1 PFU per cell. Serialdilutions of test compounds were added to cells in a final concentrationof 0.5% DMSO in growth medium. Each dilution was tested in triplicate.After three days, cell cultures (cell monolayers and supernatants) werelysed by three freeze-thaw cycles, and virus yield was quantified byplaque assay. Briefly, MDBK cells were seeded onto 6-well plates (5×105cells per well) 24 h before use. Cells were inoculated with 0.2 mL oftest lysates for 1 hour, washed and overlaid with 0.5% agarose in growthmedium. After 3 days, cell monolayers were fixed with 3.5% formaldehydeand stained with 1% crystal violet (w/v in 50% ethanol) to visualizeplaques. The plaques were counted to determine the concentration toobtain a 6-log reduction in viral load.

Studies on the antiviral activity of mCyd in cultured cells wereconducted. The primary assay used to determine mCyd antiviral potencywas a BVDV-based cell-protection assay (CPA). This assay measures theability of mCyd to protect growing MDBK bovine kidney cells fromdestruction by a cytopathic NADL strain of BVDV. The cytotoxicity of thetest drug on uninfected cells was measured in parallel. The antiviralactivities of mCyd and ribavirin in the CPA are compared in Table 8a.mCyd effectively protected de novo-infected MDBK cells in aconcentration-dependent manner with an EC₅₀=0.67±0.22 μM (Table 8a).mCyd afforded complete cytoprotection at concentrations well below theCC₅₀ for mCyd in this assay (38±9 μM). In the CPA, as well as in otherassays described below, ribavirin showed no clear antiviral effect:significant (50% or more) cell protection was not achieved in mostassays as the cytotoxicity of ribavirin overlapped and masked theprotective effect. Thus, ribavirin gave a CC₅₀ of 4.3±0.6 μM and anEC₅₀>4.3 μM in the CPA.

TABLE 8a In Vitro Activity of mCyd Against BVDV in the Cell ProtectionAssay Compound n EC₅₀, μM CC₅₀, μM mCyd 11 0.67 ± 0.22 38 ± 9 RBV 3 >4.3 4.3 ± 0.6

TABLE 8b CC₅₀ Test Results for β-D-2′-C-methyl-cytidine (Compound G),3′-O-valinyl ester of β-D-2′-C-methyl-cytidine dihydrochloride salt(Compound M), and β-D-2′-C-methyl-uracil (Compound N) Compound CC₅₀ BVDVYFV DENV 2 WNV CVB-2 Sb-1 REO G 34 2.3 54 95 80 12 11.5 13 M 24 5.882 >100 82 12 14 22 N >100 18 100 > or =100 80 >100 55 >100 Note: Celllines utilized include MT-4 for HIV; Vero 76, African green monkeykidney cells for SARS; BHK for Bovine Viral Diarrhea Virus; Sb-1 forpoliovirus Sabin type-1; CVB-2, CVB-3, CVB-4, and CVA-9 forCoxsackieviruses B-2, B-3, B-4 and A-9; and REO-1 for double-strandedRNA viruses.

TABLE 8c CC₅₀ and EC₅₀ Test Results for β-D-2′-C-methyl-cytidine(Compound G) CC₅₀ CC₅₀ CC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ Compound MT-4Vero 76 BHK Sb-1 CVB-2 CVB-3 CVB-4 CVA-9 REO-1 G 34 >100 >100 6 11 9 1326 13 Note: Cell lines utilized include MT-4 for HIV; Vero 76, Africangreen monkey kidney cells for SARS; BHK for Bovine Viral Diarrhea Virus;Sb-1 for poliovirus Sabin type-1; CVB-2, CVB-3, CVB-4, and CVA-9 forCoxsackieviruses B-2, B-3, B-4 and A-9; and REO-1 for double-strandedRNA viruses.

The overall antiviral potency of mCyd was determined against differentstrains of BVDV and both cytopathic (cp) and noncytopathic (ncp)biotypes in cell protection assays as well as in plaque reduction andyield reduction assays. The latter assays measure the output ofinfectious virus from cells and hence provide a stringent test ofantiviral efficacy. The different data sets from all three assays showagreement as summarized in Table 9. The range of 50% and 90% effectiveinhibitory concentration (EC₅₀ and EC₉₀) values for mCyd was 0.3 to 2.8μM and 0.87 to 4.8 μM, respectively.

In the BVDV yield reduction assay, subcytotoxic concentrations (circa 20μM) of mCyd suppressed de novo BVDV production by up to 6 log₁₀, to thepoint where no infectious virus was detected. A 4 log₁₀ effectivereduction in BVDV production (EC_(4 log 10) or EC₉₉₋₉₉) was attainedbetween 6.0 and 13.9 μM mCyd. In contrast, interferon alpha 2b (IFNα2b), although active against BVDV in this assay (EC₅₀ 2.6 IU per ml),never gave more than 2 logs of viral reduction, even at 1000 IU per ml.Thus, the antiviral effect of mCyd against BVDV was much greater thanthat of IFNα2b or RBV.

EXAMPLE 6 In Vitro Antiviral Activity Against Other Positive-Strand RNAViruses

mCyd has been tested for efficacy against positive-strand RNA virusesother than BVDV. Data obtained are summarized in Table 9 and 10. Againstflaviruses, mCyd showed modest activity. The composite EC₅₀ ranges (inμM) determined from both sites were: West Nile virus (46-97); YellowFever virus (9-80); and Dengue virus (59-95). For mCyd against the alphavirus, Venezuelan equine encephalitis virus, EC₅₀ values were 1.3-45 μM.mCyd was broadly active against picornaviruses, such as polio virus(EC₅₀=6 μM), coxsackie virus (EC₅₀=15 μM), rhinovirus types 5 and 14(EC₅₀s=<0.1 and 0.6 μg/ml) and rhinovirus type 2 (EC₅₀ 2-10 μM). mCydwas generally inactive against all RNA and DNA viruses tested except forthe positive-strand RNA viruses. mCyd was also found to have no activityagainst HIV in MT-4 human T lymphocyte cells or HBV in HepG2.2.15 cells.

TABLE 9 In Vitro Antiviral Activity of mCyd Against Plus-Strand RNAViruses Method of Virus Cell Antiviral Efficacy (μM) Assay Type Type nEC₅₀ EC₉₀ EC_(4 log) Cell Protection BVDV NADL cp MDBK 11 0.67 ± 0.22Assay Yield Reduction BVDV NADL cp MDBK 3 2.77 ± 1.16  4.8 ± 1.55 13.9 ±3.07 Assay BVDV MDBK 6 0.30 ± 0.07 0.87 ± 0.18 6.03 ± 1.41 New York-1ncp BVDV I-NADL cp MDBK 1   0.68 1.73 8.22 BVDV I-N-dlns ncp MDBK 1  0.59 1.49 7.14 Plaque Reduction BVDV NADL cp MDBK 3 2.57 ± 0.35 4.63 ±0.72 Assay Cell Protection West Nile Virus BHK 3 63-97 Assay CellProtection Yellow Fever BHK 1 60-80 Assay Virus 17D DENV-2 BHK 2 95 CellProtection DENV-4 BHK 1 59 Assay Polio Virus Plaque Reduction Sb-1 VERO1  6 Assay Plaque Reduction Coxsackie Virus B2 VERO 1 15 Assay cp,cytopathic virus; ncp noncytopathic virus 1-NADL cp and 1-N-dlns ncprepresent recombinant BVDV viruses

TABLE 10 In Vitro Antiviral Activity. Selectivity, and Cytotoxicity ofmCyd Virus (Cell line)^(a) EC₅₀ ^(b) (μM) CC₅₀ ^(c) (μM) WNV (Vero) 46114-124 YFV (Vero) 9-30  150->200 VEE (Veto) 1.3-45  >200 HSV-1(HFF)^(d) >100 >100 HSV-2 (HFF)^(d) >100 >100 VZV (HFF)^(d) >20 67.8 EBV(Daudi)^(d) 25.5 >50 HCMV (HFF)^(d) 9.9-15.6 67-73 MCMV (MEF) >0.8 2.4Influenza A/H1N1 (MDCK) >200 >200 Influenza A/H3N2 (MDCK) >20 45-65Influenza B (MDCK) >200  55-140 Adenovirus type 1 (A549) >200 >200Parainfluenzatype 3 (MA-104) >200 >200 Rhinovirus type 2 (KB) 2-10 >200Rhinovirus type 5 (KB)^(d) 0.6 20-30 Rhinovirus type 14 (HeLa-Ohio)^(d)<0.1  20->100 RSV type A (MA-104) >200 200 Punta Toro A(LLC-MK2) >200 >200 ^(a)HFF, human foreskin fibroblast; Daudi, Burkitt'sB-cell lymphoma; MDCK, canine kidney cells; CV-1, African green monkeykidney cells; KB, human nasopharyngeal carcinoma; MA-104, Rhesus monkeykidney cells; LLC-MK2, Rhesus monkey kidney cells; A549, Human lungcarcinoma cells; MEF, mouse embryo fibroblast; Vero, African greenmonkey kidney cells; HeLa, human cervical adenocarcinoma cells. ^(b)EC₅₀= 50% effective concentration. ^(c)CC₅₀ = 50% cytotoxic concentration.^(d)Result presented in μg/mL rather than μM.

EXAMPLE 7 Multiplicity of Infection (MOI) and Antiviral Efficacy

The cell protection assay format was used to test the effect ofincreasing the amount of BVDV virus on the EC₅₀ of mCyd. Increasing themultiplicity of infection (MOI) of BVDV in this assay from 0.04 to 0.16,caused the EC₅₀ of mCyd to increase linearly from 0.5 μM toapproximately 2.2 μM.

EXAMPLE 8 Viral Rebound in mCyd Treated Cells

The effect of discontinuing treatment with mCyd was tested in MDBK cellspersistently infected with a noncytopathic strain (strain I-N-dIns) ofBVDV. Upon passaging in cell culture, these cells continuously produceanywhere from 10⁶ to >10⁷ infectious virus particles per ml of media.This virus can be measured by adding culture supernatants from treatedMDBK (BVDV) cells to uninfected MDBK cells and counting the number ofresultant viral foci after disclosure by immunostaining with aBVDV-specific antibody. Treatment of a persistently infected cell linewith 4 μM mCyd for one cell passage (3 days) reduced the BVDV titer byapproximately 3 log₁₀ from pretreatment and control cell levels of justunder 10⁷ infectious units per ml. At this point, mCyd treatment wasdiscontinued. Within a single passage, BVDV titers rebounded tountreated control levels of just over 10⁷ infectious units per ml.

EXAMPLE 9 Mechanism of Action

In standard BVDV CPA assays, mCyd treatment results in a marked increasein total cellular RNA content as cells grow, protected from thecytopathic effects of BVDV. This is coupled with a marked decrease inthe production of BVDV RNA due to mCyd. Conversely, in the absence ofmCyd, total cellular RNA actually decreases as BVDV RNA rises due to thedestruction of the cells by the cytopathic virus. To further test theeffect of mCyd on viral and cellular RNAs, the accumulation ofintracellular BVDV RNA was monitored in MDBK cells 18-hours postinfection (after approximately one cycle of virus replication) usingReal Time RT-PCR. In parallel, a cellular housekeeping ribosomal proteinmRNA (rig S15 mRNA) was also quantitated by RT-PCR using specificprimers. The results showed that mCyd dramatically reduced BVDV RNAlevels in de novo-infected MDBK cells with an EC₅₀ of 1.7 μM and an EC₉₀of 2.3 μM. The maximum viral RNA reduction was 4 log₁₀ at the highestinhibitor concentration tested (125 μM). No effect on the level of therig S15 cellular mRNA control was observed. Together, the precedingfindings suggest that mCyd inhibited BVDV by specifically interferingwith viral genome RNA synthesis without impacting cellular RNA content.This idea is further supported by the observation (Table 4a) thatinhibition of RNA synthesis as measured by [³H]-uridine uptake in HepG2cells requires high concentrations of mCyd EC₅₀=186 μM).

In in vitro studies using purified BVDV NS5B RNA-dependent RNApolymerase (Kao, C. C., A. M. Del Vecchio, et al. (1999). “De novoinitiation of RNA synthesis by a recombinant flaviviridae RNA-dependentRNA polymerase.” Virology 253(1): 1-7) and synthetic RNA templates,mCyd-TP inhibited RNA synthesis with an IC₅₀ of 0.74 μM and was acompetitive inhibitor of BVDV NS5B RNA-dependent RNA polymerase withrespect to the natural CTP substrate. The inhibition constant (K_(i))for mCyd-TP was 0.16 μM and the Michaelis-Menten constant (K_(m)) forCTP was 0.03 μM. Inhibition of RNA synthesis by mCyd-TP required thepresence of a cognate G residue in the RNA template. The effect ofmCyd-TP on RNA synthesis in the absence of CTP was investigated in moredetail using a series of short (21 mer) synthetic RNA templatescontaining a single G residue, which was moved progressively along thetemplate. Analysis of the newly synthesized transcripts generated fromthese templates in the presence of mCyd-TP revealed that RNA elongationcontinued only as far as the G residue, then stopped (FIG. 2). Intemplates containing more than one G residue, RNA synthesis stopped atthe first G residue encountered by the polymerase. These data stronglysuggest that m-Cyd-TP is acting as a non-obligate chain terminator. Themechanism of this apparent chain termination is under furtherinvestigation.

EXAMPLE 10 Eradication of a Persistent BVDV Infection

The ability of mCyd to eradicate a viral infection was tested in MDBKcells persistently infected with a noncytopathic strain of BVDV (strainI-N-dIns). (Vassilev, V. B. and R. O. Donis Virus Res 2000 69(2):95-107.) Compared to untreated cells, treatment of persistently infectedcells with 16 μM mCyd reduced virus production from more than 6 logs ofvirus per ml to undetectable levels within two cell passages (3 to 4days per passage). No further virus production was seen upon continuedtreatment with mCyd through passage 12. At passages 8, 9 and 10 (arrows,FIG. 3), a portion of cells was cultured for two further passages in theabsence of drug to give enough time for mCyd-TP to decay and virusreplication to resume. The culture media from the cells were repeatedlytested for the re-emergence of virus by adding culture supernatants fromtreated MDBK (BVDV) cells to uninfected MDBK cells and counting theresultant viral foci after disclosure by immunostaining with aBVDV-specific antibody. Although this assay can detect a single virusparticle, no virus emerged from the cells post drug treatment. Thus,treatment with mCyd for 8 or more passages was sufficient to eliminatevirus from the persistently infected cells.

EXAMPLE 11 Combination Studies with Interferon Alpha 2B

The first study, performed in MDBK cells persistently infected with theNew York-1 (NY-1) strain of BVDV, compared the effect of monotherapywith either mCyd (8 μM) or interferon alpha 2b (200 IU/ml), or the twodrugs in combination (FIG. 4A). In this experiment, 8 μM mCyd alonereduced viral titers by approximately 3.5 log₁₀ after one passage to alevel that was maintained for two more passages. Interferon alpha 2balone was essentially inactive against persistent BVDV infection(approximately 0.1 log₁₀ reduction in virus titer) despite being activeagainst de novo BVDV infection. However, the combination of mCyd plusinterferon alpha 2b reduced virus to undetectable levels by the secondpassage and clearly showed better efficacy to either monotherapy.

In a follow up study (FIG. 4B) of MDBK cells persistently infected withthe I-N-dIns noncytopathic strain of BVDV, mCyd was supplied at fixeddoses of 0, 2, 4 and 8 μM, while interferon alpha 2b was titrated from 0to 2,000 IU per ml. Again, interferon alpha 2b was essentially inactive(0.1 log reduction in viral titer), while mCyd alone inhibited BVDV(strain I-N-dIns) propagation in a dose-dependent manner. mCyd at 8 μMreduced virus production by 6.2 log₁₀, to almost background levels.

EXAMPLE 12 Resistance Development

In early cell culture studies, repeated passaging of a cytopathic strainof BVDV in MDBK cells in the presence of mCyd failed to generateresistant mutants, suggesting that the isolation of mCyd-resistant BVDVmutants is difficult. However, studies in cell lines persistentlyinfected with noncytopathic forms of BVDV led to the selection ofresistant virus upon relatively prolonged treatment with mCyd atsuboptimal therapeutic concentrations of drug (2 to 8 μM, depending onthe experiment). In the representative experiment shown in FIG. 5A, thevirus was no longer detectable after two passages in the presence of 8μM mCyd, but re-emerged by passage 6. The lower titer of the re-emergentvirus is apparent from the data: resistant virus typically has a 10 foldor more lower titer than the wild-type virus and is easily suppressed byco-therapy with IntronA (FIG. 5A). The phenotype of the virus thatre-emerged was remarkably different from the initial wild-type virus: asshown in FIG. 5B, it yielded much smaller foci (typically, 3 to 10 timessmaller in diameter then those of the wild-type virus). This phenotypedid not change after prolonged passaging in culture in the presence ofthe inhibitor (at least 72 days), however, it quickly reverted to thewild-type phenotype (large foci) after the discontinuation of thetreatment.

RT-PCR sequencing of the resistant mutant was used to identify themutation responsible for resistance. Sequencing efforts were focused onthe NS5B RNA-dependent RNA polymerase region of BVDV, which was assumedto be the likely target for a nucleoside inhibitor. A specific S405Tamino-acid substitution was identified at the start of the highlyconserved B domain motif of the polymerase. The B domain is part of thepolymerase active site and is thought to be involved in nucleosidebinding (Lesburg, C. A., M. B. Cable, et al. Nature Structural Biology1999 6(10): 937-43). Resistance to nucleosides has been mapped to thisdomain for other viruses such as HBV (Ono et al, J Clin Invest. 2001February; 107(4):449-55). To confirm that this mutation was responsiblefor the observed resistance, the mutation was reintroduced into thebackbone of a recombinant molecular clone of BVDV. The resulting clonewas indistinguishable in phenotypic properties from the isolated mutantvirus, confirming that the S405T mutation is responsible for resistanceand that the NS5B RNA-dependent RNA polymerase is the molecular targetfor mCyd. The highly conserved nature of this motif at the nucleotidesequence (Lai, V. C., C. C. Kao, et al. J Virol 1999 73(12): 10129-36)and structural level among positive-strand RNA viruses (including HCV)allows a prediction that the equivalent mutation in the HCV NS5BRNA-dependent RNA polymerase would likely be S282T.

S405T mutant BVDV was refractory to mCyd up to the highestconcentrations that could be tested (EC₅₀>32 μM), but was alsosignificantly impaired in viability compared to wild-type virus. Asnoted above, the S405T mutant exhibited a 1-2 log₁₀ lower titer thanwild-type BVDV and produced much smaller viral plaques. In addition, themutant virus showed a marked reduction in the rate of a single cycle ofreplication (>1000-fold lower virus titer at 12 h), and accumulated toabout 100 fold lower levels than the wild-type virus even after 36 h ofreplication (FIG. 5C). The virus also quickly reverted to wild-typevirus upon drug withdrawal. Finally, the mutant was also more sensitive(˜40 fold) to treatment with IFN alpha 2b than wild-type as shown inFIG. 5D.

A second, additional mutation, C446S, was observed upon furtherpassaging of the S405T mutant virus in the presence of drug. Thismutation occurs immediately prior to the essential GDD motif in the Cdomain of BVDV NS5B RNA-dependent RNA polymerase. Preliminary studiessuggest that a virus bearing both mutations does not replicatesignificantly better than the S405T mutant, hence the contribution ofthis mutation to viral fitness remains unclear.

EXAMPLE 13 In Vivo Antiviral Activity of Val-mCyd in an Animal EfficacyModel

Chimpanzees chronically infected with HCV are the most widely acceptedanimal model of HCV infection in human patients (Lanford, R. E., C.Bigger, et al. Ilar J 2001 42(2): 117-26; Grakoui, A., H. L. Hanson, etal. Hepatology 2001 33(3): 489-95). A single in vivo study of the oraladministration of val-mCyd in the chimpanzee model of chronic hepatitisC virus infection has been conducted.

HCV genotyping on the five chimpanzees was performed by the SouthwestFoundation Primate Center as part of their mandated internal Health andMaintenance Program, designed to ascertain the disease status of allanimals in the facility to identify potential safety hazards toemployees. The five chimpanzees used in this study exhibited a high HCVtiter in a genotyping RT PCR assay that distinguishes genotype 1 HCVfrom all other genotypes, but does not distinguish genotype 1a from 1b.This indicated that the chimpanzees used in this study were infectedwith genotype 1 HCV (HCV-1).

TABLE 11 Summary of Val-mCyd In Vivo Activity Study in the ChimpanzeeModel of Chronic HCV Infection Val-mCyd Doses Frequency/Route of StudyDescription Species (N) (mg/kg) (n) Administration Study EndpointsOne-week antiviral activity Chimpanzee (5) 10 and 20 (2 each)[equivalent QD ×7 days (PO) Serum HCV RNA, serum of mCyd in chronicallyto 8.3 and 16.6 mpk of freebase], chemistries, CBCs, hepatitis C virus(genotype 1)- and vehicle control (1) general well being, infectedchimpanzees and clinical observationsSeven-Day Antiviral Activity Study in the Chimpanzee Model of ChronicHepatitis C Virus Infection

Four chimpanzees (2 animals per dose group at 10 mg/kg/day or 20mg/kg/day) received val-mCyd dihydrochloride, freshly dissolved in aflavorful fruit drink vehicle. These doses were equivalent to 8.3 and16.6 mg/kg/day of the val-mCyd free base, respectively. A fifth animaldosed with vehicle alone provided a placebo control. The study designincluded three pretreatment bleeds to establish the baseline fluctuationof viral load and three bleeds during the one week of treatment (on days2, 5 and 7 of therapy) to evaluate antiviral efficacy. The analysis wascompleted at the end of the one-week dosing period, with no furtherfollow up.

HCV RNA Determination

Serum levels of HCV RNA throughout the study were determinedindependently by two clinical hospital laboratories. HCV RNA was assayedusing a quantitative RT-PCR nucleic acid amplification test (RocheAmplicor HCV Monitor Test, version 2.0). This assay has a lower limit ofdetection (LLOD) of 600 IU/mL and a linear range of 600-850,000 IU/mL.

To aid in interpretation of the viral load declines seen during therapy,emphasis was placed on determining (i) the extent of fluctuations inbaseline HCV viral load in individual animals, and (ii) the inherentvariability and reproducibility of the HCV viral load assay. To addressthese issues, full viral load data sets obtained from the twolaboratories were compared. The results from both sites were found to beclosely comparable and affirmed both the stability of the pretreatmentHCV viral loads as well as the reliability of the HCV Roche Amplicorassay. To present the most balanced view of the study, the mean valuesderived by combining both data sets were used to generate the resultspresented in FIGS. 6 and 7. FIG. 6 presents the averaged data for dosecohorts, while FIG. 7 presents the individual animal data. The changesin viral load from baseline seen during therapy for each animal at eachsite are also summarized in Table 12.

The HCV viral load analysis from the two sites revealed thatpretreatment HCV viral loads were (i) very similar among all fiveanimals and all 3 dose groups, and (ii) very stable over the 3-weekpretreatment period. The mean pretreatment log₁₀ viral load and standarddeviations among the five individual animals were 5.8±0.1 (site 1) and5.6±0.1 (site 2). These data indicated that the c.v. (coefficient ofvariance) of the assay is only around 2% at both sites. The largestfluctuation in HCV viral load seen in any animal during pretreatment wasapproximately 0.3 log₁₀.

As seen in FIGS. 6 and 7, once a day oral delivery of val-mCyd produceda rapid antiviral effect that was not seen for the placebo animal, norduring the pretreatment period. Viral titers were substantially reducedfrom baseline after two days of therapy for all animals receivingval-mCyd, and tended to fall further under continued therapy in the twotreatment arms. By the end of treatment (day 7), the mean reductionsfrom baseline HCV viral load were 0.83 log₁₀ and 1.05 log₁₀ for the 8.3and 16.6 mg/kg/day dose groups, respectively. The titer of the placeboanimal remained essentially unchanged from baseline during the therapyperiod.

An analysis of the data from the two quantification sites on the changesin baseline HCV viral load in response to therapy is presented in Table12. Overall, the two data sets agree well, confirming the reliability ofthe assay. With the exception of animal 501, the difference in viralload between the two sites was generally 0.3 log₁₀ or less, similar tothe fluctuation observed during the pretreatment period. For animal 501,the discrepancy was closer to 0.5 log₁₀. The viral load drop seen inresponse to therapy varied from 0.436 (animal 501, site 1) to 1.514log₁₀ (animal 497, site 2). The latter corresponds to a change in HCVviral load from 535,000 (pretreatment) to 16,500 (day 7) genomes per ml.

TABLE 12 Summary of Changes in Baseline Log₁₀ HCV RNA Viral Load DuringTherapy Dose (mpk) Animal ID Site Day 2 Day 5 Day 7 0 499 1 −0.00041−0.11518 0.14085 2 −0.06604 0.10612 −0.16273 8.3 500 1 −1.15634 −0.40385−0.80507 2 −1.07902 −0.55027 −1.06259 8.3 501 I −0.25180 −0.36179−0.43610 2 −0.45201 −0.71254 −0.90034 16.6 497 1 −0.72148 −0.90704−1.27723 2 −0.85561 −1.01993 −1.51351 16.6 498 1 −0.29472 −0.28139−0.60304 2 −0.65846 −0.55966 −0.69138Exposure of Chimpanzees to mCyd

Limited HPLC analyses were performed to determine the concentration ofmCyd attained in the sera of chimpanzees following dosing with val-mCyd.In sera drawn 1 to 2 hours post dose on days 2 and 5 of dosing, mCydlevels were typically between 2.9 and 12.1 μM (750 and 3100 ng/mL,respectively) in treated animals. No mCyd was detected in pretreatmentsera or in the placebo control sera. Within 24 hours of the final dose,serum levels of mCyd had fallen to 0.2 to 0.4 μM (50 and 100 ng/mL,respectively). No mUrd was detected in any sera samples although themethodology used has a lower limit of quantification of 0.4 μM (100ng/mL) for mUrd.

Safety of mCyd in the Chimpanzee Model of Chronic HCV Infection

Chimpanzees were monitored by trained veterinarians throughout the studyfor weight loss, temperature, appetite, and general well being, as wellas for blood chemistry profile and CBCs. No adverse events due to drugwere noted. The drug appeared to be well tolerated by all four treatedanimals. All five animals lost some weight during the study and showedsome aspartate aminotransferase (AST) elevations, but these are normaloccurrences related to sedation procedures used, rather than study drug.A single animal experienced an alanine aminotransferase (ALT) flare inthe pretreatment period prior to the start of dosing, but the ALT levelsdiminished during treatment. Thus, this isolated ALT event was notattributable to drug.

EXAMPLE 14 In Vitro Metabolism

Studies were conducted to determine the stability of val-mCyd and mCydin human plasma. Val-mCyd was incubated in human plasma at 0, 21 or 37°C. and samples analyzed at various time points up to 10 hours (FIG. 8).At 37° C., val-mCyd was effectively converted to mCyd, with only 2% ofthe input val-mCyd remaining after 10 hours. The in vitro half-life ofval-mCyd in human plasma at 37° C. was 1.81 hours. In studies of the invitro stability of mCyd in human plasma, or upon treatment with a crudepreparation enriched in human cytidine/deoxycytidine deaminase enzymes,mCyd remained essentially unchanged and no deamination to the uridinederivative of mCyd (mUrd) occurred after incubation at 37° C. Only inrhesus and cynomologus monkey plasma was limited deamination observed.Incubation of mCyd at 37° C. in cynomologus monkey plasma yielded 6.7and 13.0% of mUrd deamination product after 24 and 48 hours,respectively, under conditions where control cytidine analogs wereextensively deaminated.

In addition to the TP derivatives of mCyd and mUrd, minor amounts ofmCyd-5′-diphosphate, mCyd-DP, roughly 10% the amount of thecorresponding TP, were seen in all three cell types. Lesser amounts ofmUrd-DP were detected only in two cell types, primary human hepatocytesand MDBK cells. No monophosphate (MP) metabolites were detected in anycell type. There was no trace of any intracellular mUrd and no evidencefor the formation of liponucleotide metabolites such as the5′-diphosphocholine species seen upon the cellular metabolism of othercytidine analogs.

FIG. 9 shows the decay profile of mCyd-TP determined following exposureof HepG2 cells to 10 μM [³M]-mCyd for 24 hours. The apparentintracellular half-life of the mCyd-TP was 13.9±2.2 hours in HepG2 cellsand 7.6±0.6 hours in MDBK cells: the data were not suitable forcalculating the half life of mUrd-TP. The long half life of mCyd-TP inhuman hepatoma cells supported the notion of once-a-day dosing forval-mCyd in clinical trials for HCV therapy. Phosphorylation of mCydoccurred in a dose-dependent manner up to 50 μM drug in all three celltypes, as shown for HepG2 cells in FIG. 9C. Other than the specificdifferences noted above, the phosphorylation pattern detected in primaryhuman hepatocytes was qualitatively similar to that obtained using HepG2or MDBK cells.

Contribution of mUrd

In addition to the intracellular active moiety, mCyd-TP, cells fromdifferent species have been shown to produce variable and lesser amountsof a second triphosphate, mUrd-TP, via deamination of intracellular mCydspecies. The activity of mUrd-TP against BVDV NS5B RNA-dependent RNApolymerase has not been tested to date but is planned. To date, datafrom exploratory cell culture studies on the antiviral efficacy andcytotoxicity of mUrd suggest that mUrd (a) is about 10-fold less potentthan mCyd against BVDV; (b) has essentially no antiviral activityagainst a wide spectrum of other viruses; and (c) is negative whentested at high concentrations in a variety of cytotoxicity tests(including bone marrow assays, mitochondrial function assays andincorporation into cellular nucleic acid). Based on these results, itappeared that the contribution of mUrd to the overall antiviral activityor cytotoxicity profile of mCyd is likely to be minor. Extensivetoxicology coverage for the mUrd metabolite of mCyd exists fromsubchronic studies conducted with val-mCyd in the monkey.

EXAMPLE 15 Cellular Pathways for Metabolic Activation

The nature of the enzyme responsible for the phosphorylation of mCyd wasinvestigated in substrate competition experiments. Cytidine (Cyd) is anatural substrate of cytosolic uridine-cytidine kinase (UCK), thepyrimidine salvage enzyme responsible for conversion of Cyd toCyd-5′-monophosphate (CMP). The intracellular phosphorylation of mCyd tomCyd-TP was reduced in the presence of cytidine or uridine in adose-dependent fashion with EC₅₀ values of 19.17±4.67 μM for cytidineand 20.92±7.10 μM for uridine. In contrast, deoxycytidine, a substratefor the enzyme deoxycytidine kinase (dCK), had little effect on theformation of mCyd-TP with an EC₅₀>100 mM. The inhibition of mCydphosphorylation by both cytidine and uridine, but not deoxycytidine,suggests that mCyd is phosphorylated by the pyrimidine salvage enzyme,uridine-cytidine kinase (Van Rompay, A. R., A. Norda, et al. MolPharmacol 2001 59(5): 1181-6). Further studies are required to confirmthe proposed role of this kinase in the activation of mCyd.

EXAMPLE 16 Pathways for the Cellular Biosynthesis of mUrd-TP

As outlined above, mUrd-TP is a minor metabolite arising to varyingextents in cells from different species. mUrd does not originate viaextracellular deamination of mCyd since mUrd was not seen in the cellmedium which also lacks any deamination activities. The cellularmetabolism data are consistent with the idea that mUrd-TP arises via thebiotransformation of intracellular mCyd species. Consideration of theknown ribonucleoside metabolic pathways suggests that the most likelyroutes involve deamination of one of two mCyd species by two distinctdeamination enzymes: either mCyd-MP by a cytidylate deaminase (such asdeoxycytidylate deaminase, dCMPD), or of mCyd by cytidine deaminase(CD). Further phosphorylation steps lead to mUrd-TP. These possibilitiesare under further investigation.

EXAMPLE 17 Clinical Evaluation of Val-mCyd

Patients who met eligibility criteria were randomized into the study atBaseline (Day 1), the first day of study drug administration. Eachdosing cohort was 12 patients, randomized in a 10:2 ratio to treatmentwith drug or matching placebo. Patients visited the study center forprotocol evaluations on Days 1, 2, 4, 8, 11, and 15. After Day 15, studydrug was stopped. Thereafter, patients attended follow-up visits on Days16, 17, 22, and 29. Pharmacokinetic sampling was performed on the firstand last days of treatment (Day 1 and Day 15) on all patients, underfasting conditions.

The antiviral effect of val-mCyd was assessed by (i) the proportion ofpatients with a ≧1.0 log₁₀ decrease from baseline in HCV RNA level atDay 15, (ii) the time to a ≧1.0 log₁₀ decrease in serum HCV RNA level,(iii) the change in HCV RNA level from Day 1 to Day 15, (iv) the changein HCV RNA level from Day 1 to Day 29, (v) the proportion of patientswho experience return to baseline in serum HCV RNA level by Day 29, and(vi) the relationship of val-mCyd dose to HCV RNA change from Day 1 toDay 15.

Clinical Pharmacokinetics of mCyd after Oral Administration ofEscalating Doses of Val-mCyd

Pharmacokinetics were evaluated over a period of 8 h after the firstdose on day 1 and after the last dose on day 15, with 24-h trough levelsmonitored on days 2, 4, 8, 11 and 16, and a 48-h trough on day 17.Plasma concentrations of mCyd, mUrd and Val-mCyd were measured by aHPLC/MS/MS methodology with a lower limit of quantitation (LOQ) at 20ng/ml.

The pharmacokinetics of mCyd was analyzed using a non-compartmentalapproach. As presented in the tables below, the principalpharmacokinetic parameters were comparable on day 1 and day 15,indicative of no plasma drug accumulation after repeated dosing. Theplasma exposure also appeared to be a linear function of dose. As shownin the tables below, principal pharmacokinetic parameters of drugexposure (Cmax and AUC) doubled as doses escalated from 50 to 100 mg.

TABLE 13 Pharmacokinetic parameters of mCyd at 50 mg C_(max) T_(max)AUC_(0-inf) t_(1/2) Parameters (ng/ml) (h) (ng/ml × h) (h) Day 1 Mean428.1 2.5 3118.7 4.1 SD 175.5 1.1 1246.4 0.6 CV % 41.0 43.2 40.0 13.8Day 15 Mean 362.7 2.2 3168.4 4.6 SD 165.7 1.0 1714.8 1.3 CV % 45.7 46.954.1 28.6

TABLE 14 Pharmacokinetic parameters of mCyd at 100 mg C_(max) T_(max)AUC_(0-inf) t_(1/2) Parameters (ng/ml) (h) (ng/ml × h) (h) Day 1 Mean982.1 2.6 6901.7 4.4 SD 453.2 1.0 2445.7 1.1 CV % 46.1 36.2 35.4 25.2Day 15 Mean 1054.7 2.0 7667.5 4.2 SD 181.0 0.0 1391.5 0.5 CV % 17.2 0.018.1 11.7

The mean day 1 and day 15 plasma kinetic profiles of mCyd at 50 and 100mg are depicted in the FIG. 10.

In summary, following oral administration of val-mCyd, the parentcompound mCyd was detectable in the plasma of HCV-infected subjects.mCyd exhibited linear plasma pharmacokinetics in these subjects acrossthe two dose levels thus far examined. There was no apparentaccumulation of mCyd in subjects' plasma following 15 days of dailydosing at the doses thus far examined.

Antiviral Activity of mCyd after Oral Administration of Escalating Dosesof Val-mCyd Starting at 50 mg/Day for 15 Days in HCV-Infected Patients

Serum HCV RNA levels were determined with the use of the Amplicor HCVMonitor™ assay v2.0 (Roche Molecular Systems, Branchburg, N.J., USA),which utilizes polymerase chain reaction (PCR) methods. The lower limitof quantification (LLOQ) with this assay was estimated to beapproximately 600 IU/mL and the upper limit of quantification (ULOQ)with this assay was estimated to be approximately 500,000 IU/mL.

Serum samples for HCV RNA were obtained at screening (Day −42 to −7) todetermine eligibility for the study. The Screening serum HCV RNA valuesmust be 5 log₁₀ IU/mL by the Amplicor HBV Monitor™ assay at the centralstudy laboratory.

During the study period, serum samples for HCV RNA were obtained atBaseline (Day 1), and at every protocol-stipulated post-Baseline studyvisit (Days 2, 4, 8, 11, 15, 16, 17, 22, and 29). Serum samples for HCVRNA were also collected during protocol-stipulated follow-up visits forpatients prematurely discontinued from the study.

The antiviral activity associated with the first two cohorts (50 and 100mg per day) in the ongoing study is summarized in the following tablesand graphs. Although the duration of dosing was short (15 days) and theinitial dose levels low, there were already apparent effects on thelevels of HCV RNA in the plasma of infected patients.

TABLE 15 Summary Statistics of HCV RNA in Log₁₀ Scale Day Treatment −1 12 4 8 11 15 16 17 22 29 Placebo N 6 5 5 4 4 4 4 4 3 4 3 Median 6.45 6.256.25 6.52 6.42 6.28 6.58 6.51 6.64 6.35 6.61 Mean 6.45 6.28 6.40 6.486.36 6.34 6.54 6.52 6.50 6.40 6.40 StdErr 0.25 0.12 0.15 0.18 0.24 0.160.11 0.19 0.31 0.23 0.30 50 mg N 10 10 10 10 10 10 10 10 10 10 10 Median6.81 6.69 6.58 6.55 6.56 6.46 6.57 6.45 6.54 6.73 6.67 Mean 6.72 6.726.60 6.56 6.62 6.47 6.57 6.57 6.54 6.64 6.71 StdErr 0.11 0.11 0.12 0.060.10 0.09 0.08 0.11 0.08 0.10 0.09 100 mg N 11 10 10 10 9 10 10 9 9 10 4Median 6.75 6.93 6.80 6.46 6.59 6.56 6.41 6.40 6.72 6.66 6.71 Mean 6.606.68 6.52 6.43 6.42 6.36 6.30 6.23 6.65 6.53 6.67 StdErr 0.16 0.24 0.230.21 0.24 0.22 0.22 0.23 0.16 0.18 0.17

TABLE 16 Summary Statistics of Change From Baseline (Day 1) in Log₁₀ HCVRNA Day Treatment 2 4 8 11 15 16 17 22 29 Placebo N Median 0.17 0.210.15 0.08 0.31 0.21 0.27 0.17 0.09 Mean 0.12 0.22 0.10 0.08 0.28 0.250.15 0.14 0.09 StdErr 0.09 0.12 0.16 0.06 0.15 0.10 0.18 0.09 0.16 50 mgN 10 10 10 10 10 10 10 10 10 Median −0.07 −0.13 −0.06 −0.26 −0.10 −0.13−0.21 −0.09 −0.04 Mean −0.13 −0.16 −0.11 −0.26 −0.15 −0.15 −0.18 −0.09−0.01 StdErr 0.05 0.07 0.05 0.06 0.08 0.05 0.07 0.06 0.10 100 mg N 10 109 10 10 9 9 10 4 Median −0.12 −0.24 −0.20 −0.28 −0.43 −0.49 −0.24 −0.19−0.12 Mean −0.16 −0.25 −0.21 −0.32 −0.38 −0.39 −0.18 −0.15 0.13 StdErr0.07 0.10 0.16 0.13 0.12 0.14 0.15 0.13 0.28FIG. 11 Depicts the Median Change from Baseline In Log₁₀ HCV RNA byVisit

The present invention is described by way of illustration in thefollowing examples. It will be understood by one of ordinary skill inthe art that these examples are in no way limiting and that variationsof detail can be made without departing from the spirit and scope of thepresent invention.

1. A method of treating a host infected with a Flaviviridae virus,comprising administering to the host an effective amount of a compoundhaving the structure of the formula:

or a pharmaceutically acceptable salt or prodrug thereof optionally in apharmaceutically acceptable carrier.
 2. The method according to claim 1,wherein the pharmaceutically acceptable salt is a hydrochloride salt. 3.The method according to claim 1, wherein the pharmaceutically acceptablesalt is a dihydrochloride salt.
 4. The method according to claim 1,further comprising administering the compound in a pharmaceuticallyacceptable carrier, diluent or excipient.
 5. The method according toclaim 1, wherein the compound is administered in combination oralternation with a second anti-viral agent.
 6. The method according toclaim 5, wherein the second antiviral agent is selected from the groupconsisting of an interferon, ribavirin, an interleukin, a NS3 proteaseinhibitor, a cysteine protease inhibitor, thiazolidine derivative,thiazolidine, benzanilide, phenan-threnequinone, a helicase inhibitor, apolymerase inhibitor, a nucleoside analogue, gliotoxin, cerulenin,antisense phosphorothioate oligodeoxynucleotides, an inhibitor ofIRES-dependent translation, and a ribozyme.
 7. The method according toclaim 5, wherein the second antiviral agent is an interferon.
 8. Themethod according to claim 7, wherein the second agent is selected fromthe group consisting of pegylated interferon alpha 2a, interferonalphacon-1, natural interferon, albuferon, interferon beta-1a, omegainterferon, interferon alpha, interferon gamma, interferon tau,interferon delta and interferon gamma-1b.
 9. The method of claim 8,wherein the second antiviral agent is interferon alpha
 2. 10. The methodof claim 1, wherein the host is a human.
 11. The method of claim 1,wherein the compound is in the form of a dosage unit.
 12. The method ofclaim 11, wherein the dosage unit contains 70 to 1400 mg of thecompound.
 13. The method of claim 11, wherein the dosage unit is atablet or capsule.
 14. The method of any one of claims 1-3, wherein thepharmaceutically acceptable carrier is suitable for oral or intravenousdelivery.
 15. The method of any one of claims 1-3, wherein the compoundis administered in substantially pure form.
 16. The method of any one ofclaims 1-3, wherein the compound is at least 90% by weight of theβ-D-isomer.
 17. The method of any one of claims 1-3, wherein thecompound is at least 95% by weight of the β-D-isomer.
 18. The method ofany one of claims 1-3, wherein the virus is hepatitis C.
 19. A methodfor treating a host infected with an RNA-dependant RNA polymerase virus,comprising administering an effective amount of the compound of theformula:

or its mono or dihydrochloride in pharmaceutically acceptable carrier,wherein the 5′-hydroxyl group is replaced with a 5′-OR, wherein R ishydrogen, phosphate; a stabilized phosphate prodrug; acyl; an aminoacid; a carbohydrate; a peptide; or other pharmaceutically acceptableleaving group which when administered in viva provides a compoundwherein R is independently H or phosphate.
 20. The method of claim 19wherein the virus is a F/aviviridae virus.
 21. The method of claim 19 or20 wherein the Flaviviridae virus is hepatitis C.
 22. The method ofclaim 19 or 20 wherein the host is a human.
 23. The method of claim 1,wherein the pharmaceutically acceptable salt is selected from tosylate,methanesulfonate, acetate, citrate, malonate, tartarate, succinate,benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate, formate,fumarate, propionate, glycolate, lactate, pyruvate, oxalate, maleate,salicyate, sulfate, sulfonate, nitrate, hydrobromate, hydrobromide,hydroiodide, and phosphoric acid salts.
 24. The method of claim 19,wherein the compound or a pharmaceutically acceptable salt thereof, isadministered in combination with a pharmaceutically acceptable carrier.25. The method of claim 24, wherein the carrier is suitable for oral orintravenous delivery.
 26. The method of claim 19, wherein the compoundis in the form of a dosage unit.
 27. The method of claim 19, wherein thecompound is administered in substantially pure form.
 28. The method ofclaim 19, wherein the compound is at least 90% by weight of theβ-D-isomer.
 29. The method of claim 19, wherein the compound is at least95% by weight of the β-D-isomer.
 30. The method of claim 11 or 26,wherein the dosage unit contains 50 mg to 1000 mg of the compound. 31.The method of claim 11 or 26, wherein the dosage unit contains 100 mg ofthe compound.
 32. The method of claim 11 or 26, wherein the dosage unitcontains 200 mg of the compound.
 33. The method of claim 11 or 26,wherein the dosage unit contains 400 mg of the compound.
 34. The methodof claim 11 or 26, wherein the dosage unit contains 800 mg of thecompound.
 35. The method of claim 11 or 26, wherein the dosage unitcontains 1000 mg of the compound.
 36. The method of claim 21 wherein thehost is a human.